ES2234662T3 - HEDGEHOG SIGNALING ROUTE MEDIATORS, COMPOSITIONS AND USES RELATED TO THEMSELVES. - Google Patents

Abstract

The present invention makes available methods and reagents for inhibiting aberrant growth states resulting from hedgehog gain-of-function by contacting the cell with a hedgehog antagonist, such as a small molecule, in a sufficient amount to aberrant growth state, e.g., to agonize a normal ptc pathway or antagonize hedgehog activity.

Description

Mediators of hedgehog signaling routes, compositions and related uses.

Background of the invention

Pattern formation is the activity through which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis, through the interaction of cell intrinsic lineages and cell extrinsic signaling. Inductive interactions are essential for the formation of embryonic patterns in the development of vertebrates from the initial establishment of the body's arrangement, to the formation of patterns of organ systems, to the generation of the various cell types during tissue differentiation ( Davidson, E., (1990) Development 108: 365-389; Gurdon, JB, (1992) Cell 68: 185-199; Jessell, TM et al ., (1992) Cell 68: 257-270). The effects of developmental cell interactions are diverse. Normally, the responding cells deviate from one cell differentiation path to another inducing the cells that differ from both the induced and uninduced states of the responding cells (inductions). Sometimes, cells induce their neighbors to differentiate themselves (homeogenetic induction); in other cases a cell inhibits its neighbors from differentiating themselves. Cellular interactions in early development can be sequential, so that an initial induction between two cell types leads to a progressive amplification of diversity. In addition, inductive interactions not only occur in embryonic cells, but also in adult cells, and can act to establish and maintain morphogenetic patterns as well as to induce differentiation (JB Gurdon (1992) Cell 68: 185-199).

Members of the Hedgehog family of signaling molecules mediate many important short and long range pattern formation processes during the development of invertebrates and vertebrates. In the fly, a single hedgehog gene regulates the formation of patterns of the imaginal and segmental disc. In contrast, in vertebrates, a family of hedgehog genes participates in the control of left-right asymmetry, polarity in the CNS, somite and limbs, organogenesis, chondrogenesis and spermatogenesis.

The first hedgehog gene was identified by genetic detection in the Drosophila melanogaster fruit fly (Nüsslein-Volhard, C. and Wieschaus, E. (1980) Nature 287, 795-801). This detection identified several mutations that affect embryonic and larval development. In 1992 and 1993, the molecular nature of the Drosophila hedgehog (hh) gene was reported (see, Lee et al ., (1992) Cell 71, 33-50) and since then, several hedgehog homologs have been isolated in various species of vertebrates Although only one hedgehog gene has been found in Drosophila and other invertebrates, multiple Hedgehog genes are present in vertebrates.

The vertebrate family of the hedgehog genes includes at least four members, for example, paralogs of the single hedgehog gene of Drosophila . Example hedgehog genes and proteins are described in PCT publications WO 95/18856 and WO 96/17924. Three of these members, referred to herein as Desert hedgehog ( Dhh ), Sonic hedgehog ( Shh ) and Indian hedgehog ( Ihh ), apparently exist in all vertebrates, including fish, birds and mammals. A fourth member, referred to herein as tiggie-winkle hedgehog ( Thh ), appears to be specific to fish. Desert hedgehog ( Dhh ) is expressed primarily in the testicles, both in mouse embryonic development and in rodents and adult humans; Indian hedgehog ( Ihh ) participates in bone development during embryogenesis and in bone formation in adults; and Shh , which as described above, mainly participates in morphogenic and neuroinductive activities. Given the critical inductive roles of hedgehog polypeptides in the development and maintenance of vertebrate organs, the identification of proteins that interact with hedgehog is of paramount importance both in the clinical and research context.

The various Hedgehog proteins consist of a signal peptide, a highly conserved N-terminal region and a more divergent C-terminal domain. In addition to the cleavage of the signal sequence in the secretion pathway (Lee, JJ et al . (1992) Cell 71: 33-50; Tabata, T. et al . (1992) Genes Dev . 2635-2645; Chang, DE et al . (1994) Development 120: 3339-3353), Hedgehog precursor proteins undergo an internal autoproteolytic cleavage that depends on the sequences conserved in the C-terminal part (Lee et al . (1994) Science 266: 1528-1537 ; Porter et al . (1995) Nature 374: 363-366). This autoscission leads to a 19 kD N-terminal peptide and a 26-28 kD C-terminal peptide (Lee et al . (1992) cited above; Tabata et al . (1992) cited above; Chang et al . (1994 ) cited above; Lee et al . (1994) cited above; Bumcrot, DA, et al . (1995) Mol. Cell. Biol . 15: 2294-2303; Porter et al . (1995) cited above; Ekker, SC et al . (1995) Curr. Biol . 5: 944-955; Lai, CJ et al . (1995) Development 121: 2349-2360). The N-terminal peptide remains strongly associated with the surface of the cells in which it was synthesized, while the C-terminal peptide can diffuse freely both in vitro and in vivo (Porter et al . (1995) Nature 374: 363; Lee et al ., (1994) cited above; Bumcrot et al . (1995) cited above; Mart ', E. et al . (1995) Development 121: 2537-2547; Roelink, H. et al . (1995) Cell 81 : 445-455). Interestingly, retention on the cell surface of the N-terminal peptide depends on self-cleavage, since a truncated form of HH encoded by an RNA that ends precisely at the normal position of internal cleavage can diffuse in vitro (Porter et al (1995) cited above) and in vivo (Porter, JA et al . (1996) Cell 86, 21-34). Biochemical studies have shown that the self-protection cleavage of the HH precursor protein continues through an internal thioester intermediate, which is subsequently cleaved into a nucleophilic substitution. It is likely that the nucleophile is a small lipophilic molecule that covalently attaches to the C-terminal end of the N-peptide (Porter et al . (1996) cited above) by binding it to the cell surface. The biological implications are profound. As a result of the binding, a high local concentration of the N-terminal Hedgehog peptide is generated on the surface of the cells that produce Hedgehog. It is this N-terminal peptide that is necessary and sufficient for Hedgehog signaling activities of short and long range in Drosophila and vertebrates (Porter et al . (1995) cited above; Ekker et al . (1995) cited above; Lai et al . (1995) cited above; Roelink, H. et al . (1995) Cell 81: 445-455; Porter et al . (1996) cited above; Fietz, MJ et al . (1995) Curr. Biol. 5: 643-651; Fan, C.-M et al . (1995) Cell 81: 457-465; Mart ', E., et al . (1995) Nature 375: 322-325; López-Martínez et al . (1995 ) Curr. Biol . 5: 791-795; Ekker, SC et al . (1995) Development 121: 2337-2347; Forbes, AJ et al . (1996) Development 122: 1125-1135).

HH has been involved in short and long range pattern formation processes at various sites during the development of Drosophila . In the establishment of segment polarity in early embryos, it has short-range effects that appear to be directly mediated, while in the formation of patterns of the imaginal discs, it induces long-range effects by inducing secondary signals.

In vertebrates, several hedgehog genes have been cloned in recent years. Of these genes, Shh has received most of the experimental attention, since it is expressed in different organizational centers that are the origins of the signals that form the patterns of surrounding tissues. Recent evidence indicates that Shh participates in these interactions.

Shh expression begins shortly after the start of gastrulation in the presumed midline mesoderm, the mouse nodule (Chang et al . (1994) cited above; Echelard, Y. et al . (1993) Cell 75: 1417-1430), the rat (Roelink, H. et al . (1994) Cell 76: 761-775) and the chicken (Riddle, RD et al . (1993) Cell 75: 1401-1416) and in the envelope in the zebrafish (Ekker et al . (1995) cited above; Krauss, S. et al . (1993) Cell 75: 1431-1444). In chicken embryos, the pattern of Shh expression in the nodule develops a left-right asymmetry, which appears to be responsible for the left-right position of the heart (Levin, M. et al . (1995) Cell 82: 803- 814).

In the CNS, Shh of the notochord and the basal plate seems to induce the fates of the ventral cells. When expressed ectopically, Shh leads to the ventralization of large regions of the midbrain and the rhombencephalon in the mouse (Echelard et al . (1993) cited above; Goodrich, LV et al . (1996) Genes Dev . 10: 301-312) , Xenopus (Roelink, H, et al . (1994) cited above, Ruiz i Altaba, A. et al . (1995) Mol. Cell. Neurosci . 6: 106-121) and the zebrafish (Ekker et al . ( 1995) cited above, Krauss et al . (1993) cited above; Hammerschmidt, M., et al . (1996) Genes Dev . 10: 647-658). In intermediate neuroectoderm explants at spinal cord levels, the Shh protein induces the development of the basal plate and motor neurons with different concentration thresholds, the basal plate at high concentrations and the motor neurons at lower concentrations (Roelink et al. . (1995) cited above; Mart ' et al . (1995) cited above; Tanabe, Y. et al . (1995) Curr. Biol . 5: 651-658). In addition, antibody blocking suggests that Shh produced by the notochord is required for induction mediated by the notochord of motor neuron fates (Mart ' et al . (1995) cited above). Therefore, the high concentration of Shh on the surface of the midline cells that produce Shh seems to explain the contact-mediated induction of the basal plate observed in vitro (Placzek, M. et al . (1993) Development 117: 205 -218), and the midline placement of the basal plate immediately above the notocorda in vivo . Lower concentrations of Shh released from the notochord and basal plate presumably induce motor neurons in the most distant ventrolateral regions, in a process that has been shown to be independent of in vitro contact (Yamada, T. et al . ( 1993) Cell 73: 673-686). In explants taken at the midbrain and forebrain levels, Shh also induces appropriate ventrolateral neuronal cell types, dopamine precursors (Heynes, M et al . (1995) Neuron 15: 35-44; Wang, MZ et al . (1995) Nature Med . 1: 1184-1188) and cholinergic (Ericson, J. et al . (1995) Cell (81: 747-756), respectively, indicating that Shh is a common inducer of the full length ventral specification of the SNC These observations raise the question of how the differential response to Shh is regulated in particular anteroposterior positions.

Shh from the midline also forms the patterns of the paraxial regions of the vertebrate embryo, of the somites in the trunk (Fan et al . (1995) cited above) and of the rostral region of the mesenchyme of the head of the somites ( Hammerschmidt et al . (1996) cited above). In chicken and mouse paraxial mesoderm explants, Shh encourages the expression of specific sclerotome markers such as Pax1 and Twist , at the expense of the dermamiotome marker, Pax3 . In addition, barrier filter experiments suggest that Shh mediates the induction of the scleratom directly, rather than by activation of a secondary signaling mechanism (Fan, C.-M. and Tessier-Lavigne, M. (1994) Cell 79, 1175- 1186).

Shh also induces myotome gene expression (Hammerschmidt et al . (1996) cited above; Johnson, RL et al . (1994) Cell 79: 1165-1173; Miinsterberg, AE et al . (1995) Genes Dev . 9: 2911-2922; Weinberg, ES et al . (1996) Development 122: 271-280; although recent experiments indicate that WNT family members, vertebrate homologs of Drosophila wingless , are required together (Miinsterberg et al . (1995) cited above) In a disconcerting way, the induction of the myotome in chickens requires higher concentrations of Shh than the induction of sclerotome markers (Münsterberg, et al . (1995) cited above), although the sclera is originated from somatic cells located much closer to the notocorda Similar results were obtained in zebrafish, where high concentrations of Hedgehog induce gene expression of the myotome marker and repress that of the sclerotome marker (Hammerschmidt et al . (1996) citation do above). However, unlike the amniots, these observations agree with the architecture of the fish embryo, since here, the myotome is the predominant and most axial component of the somites. Therefore, the modulation of Shh signaling and the acquisition of new signaling factors may have modified the structure of somites during vertebrate evolution.

In vertebrae limb buds, a subset of posterior mesenchymal cells, the "Polarizing Activity Zone" (ZPA), regulates the identity of the anteroposterior finger (reviewed in Honig, LS (1981) Nature 291: 72-73) . The ectopic expression of Shh or the application of pearls soaked in Shh peptide mimics the effect of grafts of anterior ZPA, generating a mirror image duplication of the fingers (Chang et al . (1994) cited above; López-Martínez et al . (1995) cited above; Riddle et al. (1993) cited above (Figure 2g). Therefore, the identity of the fingers seems to depend mainly on the concentration of Shh , although it is possible that other signals may transmit this information along of substantial distances that appear to be required by the formation of AP patterns (100-150 µm).

Similar to the interaction of HH and DPP in the imaginary discs of Drosophila , Shh in vertebrae limb buds activates the expression of Bmp2 (Francis, PH et al . (1994) Development 120: 209-218), a dpp counterpart. However, unlike DPP in Drosophila , Bmp2 does not mimic the polarizing effect of Shh in ectopic application on the tips of chicken limbs (Francis et al . (1994) cited above). In addition to the formation of the anteroposterior pattern, Shh also appears to participate in the regulation of proximal external growth of the extremities, inducing the synthesis of the FGF4 fibroblast growth factor in the posterior apical ectodermal flange (Laufer, E. et al . (1994) Cell 79: 993-1003; Niswander, L. et al . (1994) Nature 371: 609-612).

It is likely that the close relationship between Hedgehog and BMP proteins has been conserved in many, but probably not all, sites of Hedgehog expression in vertebrates. For example, in the posterior chicken intestine, Shh has been shown to induce the expression of Bmp4 , another homologue of dpp in vertebrates (Roberts, DJ et al . (1995) Development 121: 3163-3174). In addition, Shh and Bmp2, 4 or 6 show a surprising correlation in their expression in the epithelial and mesenchymal cells of the stomach, the genitourinary system, the lung, the yolks of the teeth and the hair follicles (Bitgood, MJ and McMahon, AP ( 1995) Dev. Biol . 172: 126-138). In addition, Ihh , one of the other two mouse Hedgehog genes, is expressed adjacent to cells that express Bmp in the intestine and in developing cartilage (Bitgood and McMahon (1995) cited above).

Recent evidence suggests a model in which Ihh plays a crucial role in the regulation of chondrogenic development (Roberts et al . (1995) cited above). During cartilage formation, chondrocytes move from a proliferative state, through a prehypertrophic intermediate state, to differentiated hypertrophic chondrocytes. Ihh is expressed in prehypertrophic chondrocytes and initiates a signaling cascade that leads to the blockage of chondrocyte differentiation. Its direct target is the perichondrium around the Ihh expression domain , which responds by the expression of Gli and Patched ( Ptc ), conserved transcriptional targets of the Hedgehog signals (see below). More likely, this leads to secondary signaling that results in the synthesis of parathyroid hormone-related protein (PTHrP) in the periarticular perichondrium. PTHrP sends itself signals back to prehypertrophic chondrocytes, blocking their subsequent differentiation. At the same time, PTHrP represses the expression of Ihh , thus forming a negative feedback cycle that modulates the rate of differentiation of chondrocytes.

Patched was originally identified in Drosophila as a segment polarity gene, one of a group of developmental genes that affect cell differentiation within individual segments that occur in a homologous series, along with the anterior-posterior axis of the embryo . See Hooper, JE et al . (1989) Cell 59: 751; and Nakano, Y. et al . (1989) Nature 341: 508. Patterns of homology in patched vertebrates suggest their involvement in the development of the neural tube, skeleton, extremities, craniofacial structure and skin.

Genetic and functional studies show that patched is part of the hedgehog signaling cascade, an evolutionarily conserved path that regulates the expression of several genes in the 3 'direction (downstream). See Perrimon, N. (1995) Cell 80: 517; and Perrimon, N. (1996) Cell 86: 513. Patched participates in the constitutive transcriptional repression of the target genes; Its effect is a secreted glycoprotein, encoded by hedgehog, or a vertebrate homologue, which induces transcriptional activation. The genes under the control of this route include members of the Wnt and TGF-beta families.

Patched proteins have two large extracellular domains, twelve transmembrane segments and several cytoplasmic segments. See Hooper, cited above; Nakano, cited above; Johnson, RL et al . (1996) Science 272: 1668; and Hahn H. et al (1996) Cell 85: 841. The biochemical role of patched in the hedgehog signaling path is unclear. However, direct interaction with the hedgehog protein (Chen, Y. et al . 81996) Cell 87: 553) has been reported and patched can participate in a hedgehog receptor complex, together with another transmembrane protein encoded by the smoothened gene . See Perrimon, cited above; and Chen, cited above.

The human homologue of patched was cloned and recently mapped for chromosome 9q22.3. See Johnson, cited above; and Hahn, cited above. This region has been implicated in basal cell nevus syndrome (BCNS), which is characterized by developmental abnormalities, including craniofacial and rib abnormalities, abnormalities of the hands and feet and spina bifida.

Sporadic tumors also demonstrated a loss of the two functional patched alleles. Of twelve tumors in which patched mutations were identified with a single chain conformational polymorphism detection assay, nine had chromosomal deletion of the second allele and the other three had inactivating mutations in both alleles (Galiani, cited above). The alterations did not occur in the corresponding germline DNA.

Most of the identified mutations resulted in premature termination codons or frame changes. Lench, NJ et al ., Hum. Genet 1997 Oct; 100 (5-6): 497-502. However, several were point mutations that led to amino acid substitutions in any of the extracellular or cytoplasmic domains. These mutation sites may indicate the functional importance of interaction with extracellular proteins or with cytoplasmic elements of the downstream signaling path.

The involvement of patched in the inhibition of gene expression and the appearance of sequential allelic deletions of patched in BCC (basal cell carcinoma) support a tumor suppression function for this gene. Their role in the regulation of gene families that are known to participate in cell signaling and intercellular communication provides a possible mechanism of tumor suppression.

EP 0 056 637 refers to derivatives of 4 (3H) -quinazolinone, to one procedure for its production and pharmaceutical compositions comprising these compounds.

A. Rao et al ., Journal of Indian Chem. Soc., Vol. 62, No. 3, March 1985, pages 234-7, deals with the synthesis and biological activities of certain derivatives of 3-aryl-4 (3H) -quinazolinones.

Singh BD et al ., Journal of Indian Chem. Soc., Vol. 46, No. 1, 1969 refers to the synthesis of imidazo [1,5-a] -quinazolones.

Twari SS et. al ., Journal Chem. Soc. Pak., 1981, 3 (4), pages 215-17, describes the synthesis of 2-hippyl-3-aryl-quinazolinones and their possible use as antifertility compounds.

Summary of the invention

The invention relates to the use of compounds of general formula (II) for the preparation of a composition pharmaceutical to inhibit hyperproliferation of a cell. The Compounds of general formula (II), are as follows:

Formula II

one

in which, to the extent that the valence and stability what allow,

R 1 and R 2, independently for each appearance, represent H, lower alkyl, aryl (substituted or not substituted), aralkyl (substituted or unsubstituted), heteroaryl (substituted or unsubstituted), or heteroaralkyl (substituted or not replaced);

L, independent for each occurrence, is absent or represents -alkyl, -alkenyl-, alkynyl-, - (CH 2) n O (CH 2) p -, - (CH 2) n NR 2 (CH 2) p -, - (CH 2) n S (CH 2) p -, - (CH 2) n alkenyl (CH 2) p -, - (CH 2) n alkynyl (CH 2) p -, -O (CH 2) n -, -NR 2 (CH 2) n - or -S (CH 2) n -;

       \ newpage
    

X is selected from -N (R 8) -, -O-, -S-, -Se- -N = N-, -ON = CH-, (R 8) N-N (R 8) -, -ON (R 8) -, a heterocycle, or a direct link between L e Y;

And select from -C (= O) -, -C (= S) -, -S (O2) -, -S (O) -, -C (= NCN) -, -P (= O) (OR_2) -, a heteroaromatic group, or a direct link between X and Z;

Z is selected from -N (R 8) -, -O-, -S-, -Se- -N = N-, -ON = CH-, R 8 N-NR 8 -, -ONR 8 -, a heterocycle, or a direct link between Y and L;

R 8, independently for each occurrence, represents H, lower alkyl, aryl (substituted or unsubstituted), aralkyl (substituted or unsubstituted), heteroaryl (substituted or not substituted), or heteroaralkyl (substituted or unsubstituted), or two R_ {8} taken together form a ring of 4 to 8 members, along with the atoms to which they are attached, ring that can include one or more carbonyls;

W represents a benzene or pyridonic ring, substituted or unsubstituted, condensed to the pyrimidone ring;

p represents independently for each appearance, an integer from 0 to 10; and n, individually for each occurrence, represents an integer from 0 to 10.

In certain embodiments, R_ {1} represents a substituted or unsubstituted aryl or heteroaryl group. In certain realizations, X-Y-Z taken together represents a urea or an amide. In certain embodiments, W is a substituted or unsubstituted benzene ring. In certain embodiments, X represents diazacarbocycle. In certain embodiments, R2 represents an aryl or heteroaryl group, substituted or unsubstituted. In certain embodiments, R 8, independently for each occurrence, it is selected from H and alkyl lower. In certain embodiments, X is selected from -N (R 8) -, -O-, -S- and a direct link; And it is selected of -C (= O) -, -C (= S) - and -S (O2) -; and Z is selected from -N (R_ {8}) -, -O-, -S- and a direct link, so that at minus one of X and Z be present. In certain embodiments, at least One of X and Z is present. In certain embodiments, and it select from -C (= O) -, -C (= S) - and -S (O2) -.

In certain embodiments, the hedgehog antagonist inhibits signal transduction mediated by hedgehog with a de_ {50} of 1 mM or less, 1 \ uM or less, 100 nM or less, 10 nM or less, 1 nM or less. The cell can contact the hedgehog antagonist in vitro or in vivo . In certain embodiments, the hedgehog antagonist is administered as part of a therapeutic or cosmetic application, such as regulation of neuronal tissues, bone and cartilage formation and repair, spermatogenesis regulation, smooth muscle regulation, regulation of the lung, liver and other organs that come from the primitive intestine, the regulation of hematopoietic function or the regulation of skin and hair growth.

In still another aspect, the invention relates to to the use of a compound that has the general structure of the Formula II:

Formula II

2

in which, to the extent that the valence and stability what allow,

R 1 and R 2, independently for each appearance, represent H, lower alkyl, - (CH 2) n -aryl (for example, substituted or unsubstituted), or - (CH 2) n -heteroaryl (for example, substituted or unsubstituted);

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L, independently for each occurrence, is absent or represents -alkyl, -alkenyl-, alkynyl-, - (CH2) nO
(CH 2) p -, - (CH 2) n NR 2 (CH 2) p -, - (CH 2) n S (CH 2) p -, - (CH 2) n alkenyl (CH 2) p -, - (CH 2) n alkynyl ( CH 2) p -, -O (CH 2) n -, -NR 2
(CH 2) n - or -S (CH 2) n -;

X is -NH-;

And select from -C (= O) -, -C (= S) -, -S (O2) -, -S (O) -, -C (= NCN) -, -P (= O) (OR_2) -, a heteroaromatic group, or a direct link between X and Z;

Z is selected from -N (R 8) -, -O-, -S-, -Se- -N = N-, -ON = CH-, R 8 N-NR 8 -, -ONR 8 -, a heterocycle, or a direct link between Y and L;

R 8, independently for each occurrence, represents H, lower alkyl, - (CH 2) n -aryl (for example, substituted or unsubstituted), - (CH 2) n -heteroaryl (for example, substituted or unsubstituted), or two R 8 taken together form a ring of 4 to 8 members, for example with X_ {1} and Z_ {1} or X_ {2} and Z_ {2}, ring that can include one or more carbonyls;

W represents a benzene or pyridonic ring, substituted or unsubstituted, condensed to the pyrimidone ring;

p represents independently for each occurrence, an integer from 0 to 10, preferably from 0 to 3; and n, individually for each occurrence, represents a number integer from 0 to 10, preferably 0 to 5.

In certain embodiments, L adjacent to X represents - (lower unbranched alkyl) -. In certain embodiments, R 1 represents an aryl or heteroaryl ring not substituted, or an aryl or heteroaryl ring substituted with substituents selected from H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, hydroxyl, (alkyl-O no branched), silyloxy, amino, nitro, thiol, amino, imino, amido, phosphoryl, phosphonate, phosphine, carbonyl, carboxyl, carboxamide, anhydride, silyl, thioether, alkyl sulfonyl, aryl sulfonyl, sulfoxide, selenoether, ketone, aldehyde, ester or - (CH 2) m -R 8.

In certain embodiments, R_ {1} represents a unsubstituted aryl or heteroaryl ring, or an aryl ring or heteroaryl substituted with substituents selected from H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, nitro, thiol, imino, amido, carbonyl, carboxyl, anhydride, thioether, alkyl sulfonyl, aryl sulfonyl, ketone, aldehyde and ester. In certain embodiments, R_ {1} represents an unsubstituted aryl or heteroaryl ring, or a ring aryl or heteroaryl substituted with substituents selected from H, halogen, cyano, alkyl, alkenyl, alkynyl, nitro, amido, carboxyl, anhydride, alkyl sulfonyl, ketone, aldehyde and ester. In certain embodiments, R2 is aryl substituted or unsubstituted or a heteroaryl ring. In certain embodiments, X-Y-Z represents a amide or urea junction. In certain embodiments, R 8 represents H For all appearances. In certain embodiments, R2 is a substituted or unsubstituted aryl or heteroaryl ring. In certain embodiments, L is absent adjacent to R_ {1}.

In certain embodiments, the compound has a selected structure of the structures represented in the Figures 32j, k and l,

3

The synthesis of the compounds used according to the invention may involve a method of preparing a compound bicyclic, which comprises heating a reaction mixture that it comprises a compound that has a structure of Formula X with a carboxylic acid anhydride according to the scheme:

4

in which W represents a ring aryl or heteroaryl, Y

R 10 represents alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or heteroaralkyl substituted or unsubstituted, and in which the anhydride has a structure of (R 10 CH 2 C = O) 2 O, in which R 'is CH 2 R 10.

In certain embodiments, the reaction mixture It consists essentially of the compound of formula X and the anhydride of carboxylic acid at the beginning of the reaction.

Synthesis may also involve a method for preparing an amine, which comprises contacting a compound which has a structure of Formula XIII with an amine that has a H2 {NR} {12} structure according to the scheme:

5

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in which W represents a ring aryl or heteroaryl:

R1 represents H, lower alkyl, - (CH 2) n -aryl or - (CH 2) n -heteroaryl;

L is absent or represents - (CH 2) n -alkyl, -alkenyl-, alkynyl-, - (CH 2) n -alkenyl-, - (CH 2) n} -alkynyl-, - (CH2) n
O (CH 2) p -, - (CH 2) n NR 2 (CH 2) p -, - (CH 2) n S (CH 2) p -, - (CH 2) n alkenyl (CH 2) p -, - (CH 2) n alkynyl (CH 2) p -, -O (CH 2) n -, -NR 2 (CH 2) n - or -S (CH 2) ) n -;

Y represents halogen;

R 10 represents alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or heteroaralkyl substituted or unsubstituted; Y

R 12 represents a lower alkyl group or a silyl group,

in which the amine and the compound that has a Structure of Formula XIII are combined with a polar solvent that It comprises less than about 50% water.

In certain embodiments, the polar solvent It includes alcohol. In certain embodiments the alcohol is selected of methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butanol, sec-butanol, ethylene glycol, and 1,3-propanediol.

Synthesis may also involve a method for preparing a compound comprising performing the steps according to the following scheme:

6

in which stage (A) comprises reacting a compound that has a structure of Formula X, wherein W represents an aryl or heteroaryl ring substituted or not substituted, such as a benzene ring, which has an amino group and a carboxylic acid group in adjacent positions (ortho), with a acylating agent having the formula R 10 CH 2 C (= O) X ', in which R 10 independently of each occurrence, represents alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or substituted or unsubstituted heteroaralkyl, and X 'represents a halogen or -OC (= O) CH 2 R 10, under conditions that produce a compound that has a formula structure XI;

step (B) comprises reacting a compound that has a structure of Formula XI with an amine that it has the formula R 1 LNH 2, in which R 1 and L are as defined above, under conditions that result in a compound having a structure of Formula XII;

step (C) comprises reacting a compound having a structure of Formula XII with an agent halogenating, such as chlorine, bromine, iodine, N-Bromosuccinimide, N-Chlorosuccinimide, N-iodosuccinimide, ClBr, IBr, ClI, or a reagent that generates a halogen radical (such as Cl; Br; or I) under conditions that result in a compound that has a structure of Formula XIII, in which Y 'represents a halogen such as Cl, Br or I;

step (D) comprises reacting a compound that have a structure of Formula XIII, with an amine having the formula H 2 NR 12; in which R_ {12} represents a lower alkyl group or a silyl group, such as a group trialkylsilyl, triarylsilyl, dialkylarylsilyl or diarylalkylsilyl, under conditions that result in a compound having a structure of Formula XIV; Y

Step (E) comprises reacting a compound having a structure of Formula XIV with a termination group having a structure R2 V 'to produce a compound having a structure of Formula XX, in which R2 } is as defined above, and V 'represents a functional group selected from ZC (= W) Cl, ZC (= W) Br, isocyanate, isothiocyanate, ZC (= W) WC (= W) ZR 2, ZSO 2 Cl, ZSO 2 Br, ZSOCl, ZSOBr, or an activated acylating moiety prepared in situ .

Synthesis may also involve a method for preparing a compound, comprising performing steps according to the scheme:

7

in which stage (A) comprises reacting a compound that has a structure of Formula XV, in which P 'represents H or a protecting group, W represents a substituted or unsubstituted aryl or heteroaryl ring, such as a benzene ring, which has an amino group and an acid group carboxylic or ester in adjacent positions (ortho), with an agent acylating agent having the formula PXLC (= O) X ', in which X and L are as defined above, P represents a protecting group, and X 'represents a halogen, -OX (= O) LXP, or a functional group generated by reacting a carboxyl group with an agent activator, such as carbodiimide (for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, etc.) phosphorus-based reagents (such as BOP-Cl, PyBROP, etc.) oxalyl chloride, phosgene, triphosgene, carbonyldiimidazole, or any other reagent that react with a carboxylic acid group resulting in a reactive intermediate that has an increased susceptibility, relative to carboxylic acid, versus coupling with an amine, under conditions that produce a compound that has a structure of Formula XVI;

step (B) comprises deprotecting the ester of a compound that has a structure of Formula XVI to produce a carboxylic acid having a structure of Formula XVII, if it were necessary;

step (C) comprises reacting a compound that has a structure of Formula XVII with an amine that it has the formula R 1 LNH 2, in which R 1 and L are as defined above, under conditions that result in a compound having a structure of Formula XVIII;

step (D) comprises removing the group protector P of a compound having a structure of Formula XVIII to generate a compound that has a Formula structure XIX;

Step (E) comprises reacting a compound having a structure of Formula XIX with a termination group having a structure R 2 Y 'to produce a compound having a structure of Formula XXI, in which R 2 } is as defined above, and Y 'represents a functional group selected from ZC (= W) Cl, ZC (= W) Br, isocyanate, isothiocyanate, ZC (= W) WC (= W) ZR 2, ZSO 2 Cl, ZSO 2 Br, ZSOCl, ZSOBr, or an activated acylating moiety prepared in situ .

Brief description of the drawings

Figures 1-31 represent useful reactions to synthesize compounds according to the present invention.

Figures 32a-1 illustrate Representative compounds according to the present invention.

Figure 33 presents the test results biological aspects of an example compound of the invention.

Figure 34 presents the effects of a compound example of the invention on lung tissue.

Detailed description of the invention I Overview

The present invention relates to the discovery that signal transduction pathways regulated by
hedgehog , patched (ptc), gli and / or smoothened can be inhibited, at least in part, by small molecules. Although it is not desired to restrict itself to any particular theory, the activation of a receptor may be the mechanism by which these agents act. For example, the ability of these agents to inhibit the proliferation of patched function loss cells (ptc lof) may be due to the ability of such molecules to interact with hedgehog , patched or smoothened , or at least to interfere with the ability of these proteins to activate a signal transduction pathway mediated by hedgehog , ptc and / or smoothened .

Therefore, it is specifically contemplated that these small molecules that interfere with aspects of the hedgehog , ptc or smoothened signal transduction activity may also inhibit proliferation (or other biological consequences) in cells that have a hedgehog function gain phenotype. In preferred embodiments, the subject inhibitors are organic molecules having a molecular weight of less than 2500 amu, more preferably less than 1500 amu and even more preferably, less than 750 amu, and can inhibit at least some of the biological activities of the proteins. hedgehog, preferably specifically in the target cells.

Therefore, the methods of the present invention include the use of small molecules that are agonists of the inhibition of ptc of hedgehog signaling in the regulation of repair and / or functional performance of a wide variety of cells, tissues and organs that They have the hedgehog function gain phenotype. For example, the object method has therapeutic and cosmetic applications that range from the regulation of neuronal tissues, the formation and repair of bone and cartilage, the regulation of spermatogenesis, the regulation of smooth muscle, the regulation of the lung, liver and tissue from other organs that come from the primitive intestine, the regulation of hematopoietic function, the regulation of skin and hair growth, etc. In addition, the subject methods can be carried out in the cells that are obtained in culture ( in vitro ), or in the cells in a complete animal ( in vivo ). See, for example, PCT publications WO 95/18856 and WO 96/17924 (whose descriptive reports are expressly incorporated by reference herein).

In a preferred embodiment, the object use may be to treat epithelial cells that have a hedgehog function gain phenotype.

In another preferred embodiment, the object use can be used as part of a treatment regimen for malignant medulloblastoma and other primitive neuroectodermal tumors CNS malignant.

In another aspect, the present invention provides pharmaceutical preparations comprising, as an active ingredient , a hedgehog antagonist or a ptc agonist, as described herein, formulated in an amount sufficient to inhibit, in vivo , the proliferation or Other biological consequences of hedgehog function gain.

Object treatments that use hedgehog antagonists can be effective for both animal and human subjects. Animal subjects to which the invention can be applied encompass domestic animals and livestock, raised both as pets or for commercial purposes. Examples are dogs, cats, cattle, horses, sheep, pigs and goats.

II. Definitions

For convenience, certain terms used in The specification, examples and appended claims are gather here.

The expression "modification or mutation aberrant "of a gene refers to genetic lesions such as, for example, deletions, substitution or addition of nucleotides to a gene, as well as gross chromosomal redispositions of the gene and / or abnormal methylation of the gene. Also, the alteration in the expression of a gene refers to aberrant levels of gene transcription in relation to the levels in a normal cell in conditions similar, as well as the splicing process of unnatural type  of the mRNA transcribed from the gene.

"Basal cell carcinomas" exist in a variety of clinical and histological forms, such as nodular-ulcerative, superficial, pigmented, Morphea type, fibroepitheliomas and nevoid syndrome. Carcinomas Basal cell are the most common cutaneous neoplasms found in humans. Most new cases of skin cancers other than melanoma are within this category.

"False wounds" refers to cases in which that large superficial areas of the skin have been removed or lost of an individual due to heat and / or chemical agents.

The term "carcinoma" refers to a new malignant growth composed of epithelial cells that tend to infiltrate surrounding tissues and lead to metastases. Examples of carcinomas include: "basal cell carcinoma," which is a skin epithelial tumor that, although rarely metastatic, has potential for local invasion and destruction, "squamous cell carcinoma," which refers to carcinomas that arise. of the squamous epithelium and having cuboid cells; "carcinosarcoma", which includes malignant tumors composed of carcinomatous and sarcomatous tissues; "adenocystic carcinoma" means carcinoma characterized by cylinders or bands of hyaline or mucinous stroma separated or surrounded by nests or cords of small epithelial cells, which occur in the mammary and salivary glands and mucous glands of the respiratory tract; "squamous cell carcinoma", which refers to cancer cells that tend to differentiate in the same way as those in the epidermis; that is, they tend to form spiny cells and undergo cornification; "nasopharyngeal carcinoma", which refers to a malignant tumor that arises in the epithelial lining of the space behind the nose; and "renal cell carcinoma," which is related to renal parenchyma carcinoma composed of tubular cells in various arrangements. Other carcinomatous epithelial growths are "papillomas", which refer to benign tumors derived from the epithelium and which have a papillomavirus as the causative agent; and "epidermoid cysts", which refer to a brain or meningeal tumor formed by the inclusion of ectodermal elements at the time of the groove closure
neural.

The "chorion" or "dermis" refers to the layer of the skin below the epidermis, which consists of a dense bed of vascular connective tissue and containing nerves and the terminal organs of the senses. Follicular roots and the sebaceous and sweat glands are structures of the epidermis that are deeply included in the dermis.

"Dental tissue" refers to the tissue in the mouth that is similar to epithelial tissue, for example, tissue of the gum The method of the present invention is useful for treating periodontal disease

"Dermal skin ulcers" refer to to skin lesions caused by superficial loss of tissue, Normally with inflammation. Dermal skin ulcers that can be treated by the method of the present invention include pressure sores, diabetic ulcers, insufficiency ulcers venous and arterial ulcers. The decubitus wounds refer to chronic ulcers that result from pressure applied to areas of the skin for prolonged periods of time. The wounds of this type are often referred to as bedsores or bedsores Pressure. Ulcers due to venous insufficiency result from stagnation of blood or other defective vein fluids. Arterial ulcers refer to necrotic skin in the area around arteries that have poor blood flow.

The term "DE_ {50}" means the dose of a drug that produces 50% of its maximum response or effect.

An "effective amount" of, for example, a hedgehog antagonist, with respect to the method being treated, refers to an amount of the antagonist in a preparation which, when applied as part of a desired dosage regimen, causes, for example , a change in the rate of cell proliferation and / or in the state of differentiation of a cell and / or in the survival rate of a cell according to clinically acceptable criteria for the disorder to be treated or for the cosmetic purpose.

The terms "epithelia", "epithelial" and "epithelium" refers to the cell lining of the internal and external body surfaces (cutaneous, mucous and serous), which include the glands and other structures derived from the same, for example, keratinocytes, esophageal cells, epidermal and epithelial hair follicle. Other fabrics Example epithelials include: olfactory epithelium, which is the epithelium pseudostratified lining the olfactory region of the cavity nasal, and containing the receptors for the sense of smell; he glandular epithelium, which refers to the epithelium composed of cells secretaries; the squamous epithelium that refers to the epithelium composed of plate-like flattened cells. The term epithelium may also refer to the transitional epithelium, such as the which is characteristically covering the hollow organs that are subject to a great mechanical change due to contraction and bloating, for example, tissue that represents a transition between the stratified squamous epithelium and the columnar.

The term "epithelialization" refers to healing by the growth of epithelial tissue on a bare surface.

The term "epidermal gland" refers to to an aggregation of cells associated with the epidermis and specialized in secreting or excreting unrelated materials with your ordinary metabolic needs. For example, the "sebaceous glands" are holocrine glands in the chorion that They secrete an oily substance and sebum. The term "glands sweat "refers to the glands that secrete sweat, located in the chorion or subcutaneous tissue, which are opened by a duct on the surface of the body.

The term "epidermis" refers to the layer outermost and non-vascularized skin, derived from ectoderm embryonic, which varies in thickness from 0.07-1.4 mm On the palmar and plantar surfaces it comprises, from the inside out, five layers: basal layer composed of cylindrical cells arranged perpendicularly; spiny or spiny cell compound layer of flattened polyhedral cells with short extensions or thorns; granular layer composed of flattened granular cells, clear layer composed of several layers of transparent cells, clear, in which the nuclei are vague or absent; and cape cornea composed of flattened, cornified and anucleated cells. In the epidermis of the general body surface, the clear coat It is normally absent.

"Excision wounds" include, tears, abrasions, cuts, punctures or lacerations in the epithelial layer of the skin and can extend to the dermal layer and even to subcutaneous fat and below. The wounds of excision may result from surgical or penetration interventions accidental skin.

The "growth state" of a cell is refers to the cell proliferation rate and / or the state of cell differentiation. A "state of growth altered "is a state of growth characterized by a rate abnormal proliferation, for example, a cell that shows a increase or decrease in the proliferation rate in relation to a normal cell

The term "hair" refers to a filiform structure, especially epidermal structure specialized compound of keratin and that develops from of a papilla sunk in the chorion, produced only by mammals and characteristic of this group of animals. In addition, "hair" It can refer to the set of such hairs. A "follicle hairy "refers to one of the tubular invaginations of the epidermis that encloses the hairs, and from which the hairs The "hair follicle epithelial cells" are refer to epithelial cells surrounding the dermal papilla in the hair follicle, for example, stem cells, sheath cells external root, matrix cells and sheath cells internal root Such cells may be normal cells not malignant, or transformed / immortalized cells.

The term " hedgehog antagonist" refers to an agent that enhances or recapitulates the bioactivity of patched , such as to suppress transcription of the target genes. Preferred hedgehog antagonists can be used to overcome a loss of ptc function and / or a smoothened function gain , these latter also being called smoothened antagonists. The term " hedgehog antagonist", as used herein, refers not only to any agent that can act by directly inhibiting the normal function of the hedgehog protein, but also to any agent that inhibits the hedgehog signaling pathway and , therefore, recapitulates the ptc function.

The term " hedgehog function gain" refers to an aberrant modification or mutation of a ptc gene, a hedgehog gene, or a smoothened gene, or a decrease (or loss) in the level of expression of such a gene, which gives as a phenotype that resembles contacting a cell with a hedgehog protein results, for example, aberrant activation of a hedgehog pathway. The gain in function may include a loss of the ability of the ptc gene product to regulate the level of expression of the Ci genes, for example, Gli1, Gli2 and Gli3 . The term " hedgehog function gain" is also used herein to refer to any similar cellular phenotype (for example, that shows excess proliferation) that occurs due to an alteration anywhere in the hedgehog signal transduction path. , including, but not limited to, a modification or mutation of the hedgehog itself. For example, a tumor cell with an abnormally high proliferation rate due to activation of the hedgehog signal path would have a "hedgehog function gain" phenotype, even if hedgehog was not mutated in that cell.

As used herein, "immortalized cells" refers to cells that have been modified by chemical and / or recombinant means, so that cells can grow through an indefinite number of crop divisions.

"Internal epithelial tissue" refers to tissue inside the body that has similar characteristics to the epidermal layer of the skin. Examples include the coating of the intestine. The method of the present invention is useful for stimulate the healing of certain internal wounds, for example, wounds that result from surgery.

The term "keratosis" refers to the Proliferative skin disorder characterized by hyperplasia of the corneal layer of the epidermis. Keratotic disorders example include follicular keratosis, palmar and plantar keratosis, Pharyngeal keratosis, keratosis pilaris and actinic keratosis.

The term "DL_ {50}" means the dose of a drug that is lethal in 50% of test subjects.

The term "nail" refers to the plate cutaneous cutaneous on the dorsal surface of the distal end of a finger or toe

The term "loss of patched function" refers to an aberrant modification or mutation of a ptc gene, or a decrease in the level of gene expression, which results in a phenotype that resembles contacting a cell with a hedgehog protein, for example, aberrant activation of a hedgehog route. Loss of function may include a loss of the ability of the ptc gene product to regulate the level of expression of the Ci genes, for example, Gli1, Gli2 and Gli3 .

A "patient" or "subject" who has to being treated by the object method can mean either a human being or a non-human animal.

The term "prodrug" is intended to encompasses compounds that, under physiological conditions, become in therapeutically active principles of the present invention. A common method of obtaining a prodrug is to include remains selected that are hydrolyzed under physiological conditions to Reveal the desired molecule. In other embodiments, the prodrug it is converted by an enzymatic activity of the animal Guest.

As used herein, "that proliferates "and" proliferation "refers to cells that They experience mitosis.

During the entirety of this application, the term "proliferative skin disorder" refers to any skin disease / disorder characterized by unwanted or aberrant proliferation of skin tissue. These states are typically characterized by cell proliferation epidermal or incomplete cell differentiation, and include, by example, ichthyosis associated with the X chromosome, psoriasis, dermatitis atopic, allergic contact dermatitis, hyperkeratosis epidermolytic and seborrheic dermatitis. For example, the epidermodisplasia is a defective form of development of the epidermis. Another example is "epidermolysis," which refers to a state of sagging epidermis with vesicle formation and blisters, either spontaneously or at the site of a trauma

As used herein, "psoriasis" refers to a skin disorder hyperproliferative that alters the regulatory mechanisms of the skin. In particular, lesions are formed that include alterations Primary and secondary epidermal proliferation responses inflammatory skin and an expression of regulatory molecules, such as lymphokines and inflammatory factors. Psoriatic skin becomes morphologically characterized by an increase in the turnover of epidermal cells, thickened epidermis, abnormal keratinization, inflammatory cell infiltrates in the dermal layer e infiltration of polymorphonuclear leukocytes in the epidermal layer, resulting in an increase in the basal cell cycle. Additionally, hyperkeratotic cells are present and parakeratotic

The term "skin" refers to the cover protective outer body, which consists of the chorion and the epidermis, and it is understood that it includes the sweat glands and sebaceous, as well as the hair follicle structures. During the the whole of the present application, the adjective can be used "cutaneous" and it should be understood that it generally refers to skin characteristics, as appropriate for the context in The one they use.

The term " smoothened function gain " refers to an aberrant modification or mutation of a smo gene, or an increase in the level of gene expression, which results in a phenotype that resembles contacting a cell with a hedgehog protein, for example, aberrant activation of a hedgehog route. Although it is not desired to restrict to any particular theory, it is noted that ptc may not signal directly in the cell, but rather interacts with smoothened , another membrane binding protein downstream of ptc in hedgehog signaling (Marigo et al . , (1996) Nature 384: 177-179). The smo gene is a segment polarity gene required for the correct pattern formation of each segment in Drosophila (Alcedo et al ., (1996) Cell 86: 221-232). Homologues have been identified in smo humans. See, for example, Stone et al . (1996) Nature 384: 129-134, and GenBank registration number U84401. The smoothened gene encodes an integral membrane protein that is characteristic of the heterotrimetric receptors associated with G proteins; that is, 7 transmembrane regions. This protein shows homology with the Frizzle (Fz) protein of Drosophila, a member of the wingless path. It was originally thought that smo codes for a receiver of the Hh signal. However, this suggestion was subsequently denied, since evidence was obtained that ptc was the Hh receptor. Smo- expressing cells do not bind Hh, indicating that smo does not interact directly with Hh (Nusse, (1996) Nature 384: 119-120). Instead, it is believed that the binding of Sonic hedgehog (SHH) to its receptor, PTC H, prevents normal inhibition by smoothened PTC H (SMO), a transmembrane space protein.

Recently, it has been reported that smoothened activating mutations occur in sporadic basal cell carcinoma, Xie et al . (1998) Nature 391: 90-2, and in primitive neuroectodermal tumors of the central nervous system. Reifengerger et al . (1998) Cancer Res 58: 1798-803.

The term "therapeutic index" refers to to the therapeutic index of a drug defined as DL_ {50} / DE_ {50}.

As used herein, "transformed cells" refers to cells that have spontaneously converted to a state of uncontrolled growth, that is, they have acquired the ability to grow in a number undefined of divisions in cultivation. Transformed cells they can be characterized by terms such as neoplastic, anaplastic and / or hyperplastic, regarding their loss of control of growth

The term "acylamino" is recognized in the technique and refers to a remainder that can be represented by the General Formula:

8

in which R_ {9} is as it is defined above and R '11 represents a hydrogen, a alkyl, an alkenyl or - (CH 2) m -R 8, in which m and R_ {8} are as defined previously.

In this document, the term "group aliphatic "refers to a linear chain hydrocarbon group, branched chain or cyclic aliphatic, and includes aliphatic groups saturated and unsaturated, such as an alkyl group, a group alkenyl, and an alkynyl group.

The terms "alkenyl" and "alkynyl" they refer to analog aliphatic groups analogous in length and the possible substitution to the alkyls described above, but containing at least one double or triple bond, respectively.

The terms "alkoxy" or "alkoxy", such as used herein, they refer to a group alkyl, as defined above, which has a radical oxygen attached to it. Representative alkoxy groups include, methoxy, ethoxy, propyloxy, tert-butoxy and Similar. An "ether" is two joined hydrocarbons covalently by oxygen. Consequently, the substituent of an alkyl that converts that alkyl into an ether is or resembles to an alkoxy, as it may be represented by one of -O-alkyl, O-alkenyl, -O-alkynyl, -O- (CH 2) m -R 8, in which m and R 8 are as defined above.

The term "alkyl" refers to the radical of saturated aliphatic groups, including chain alkyl groups linear, branched chain alkyl groups, cycloalkyl groups (alicyclic), cycloalkyl groups alkyl-substituted and alkyl groups cycloalkyl substituted. In the realizations preferred, a straight chain or branched chain alkyl has 30 carbon atoms or less in the skeleton (for example, C_ {1} -C_ {30} for linear chains, C 3 -C 30 for branched chains), and more preferably 20 or less. Also, the cycloalkyls Preferred have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

In addition, the term "alkyl" (or "alkyl lower "), as used throughout the entire memory descriptive, examples and claims, intended to include both "unsubstituted alkyls" such as "alkyls substituted ", the latter referring to alkyl residues that they have substituents that replace a hydrogen in one or more of the carbons of the hydrocarbon skeleton. Such substituents they can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate or a  thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl or an aromatic or heteroaromatic moiety. Those skilled in the art will understand that the remains substituted in the hydrocarbon chain can be substituted themselves, if It is appropriate. For example, the substituents of an alkyl substituted may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and  sulfonate), and silyl groups, as well as ethers, alkylthio, carbonyls (including ketones, aldehydes, carboxylates and esters), -CF_3, -CN and the like. Alkyl substituted example is described later. The cycloalkyls may be substituted additionally with alkyls, alkenyl, alkoxyls, alkylthio, aminoalkyl, carbonyl substituted alkyls, -CF_3, -CN and the like.

Unless the number of carbons is specified otherwise, "lower alkyl", as used in the This document means an alkyl group, as defined previously, but that has from one to ten carbons, more preferably from one to six carbon atoms in its skeleton structure Also, "lower alkenyl" and "lower alkynyl" have similar chain lengths. During the entire application, preferred alkyl groups They are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.

The term "alkylthio" refers to a group alkyl, as defined above, which has a radical of sulfur attached to it. In preferred embodiments, the rest of "alkylthio" is represented by one of - S-alkyl, -S-alkenyl, -S-alkynyl and -S- (CH 2) m -R 8, in which m and R 8 are as defined above. Alkylthio groups Representative include methylthio, ethylthio, and the like.

The terms "amine" and "amino" are recognized in the art and refer to both substituted amines as unsubstituted, for example, a remainder that can be represented by the general formula:

9

in which R_ {9}, R_ {10} and R '10 each independently represents a hydrogen, a alkyl, an alkenyl, - (CH 2) m -R 8, or R 9 and R 10 taken together with the N atom to which they are attached complete a heterocycle that has from 4 to 8 atoms in the ring structure; R 8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or a integer in the range of 1 to 8. In the embodiments preferred, only one of R 9 or R 10 can be a carbonyl, for example, R 9, R 10 and nitrogen together do not form a imida Even in the most preferred embodiments, R 9 and R 10 (and optionally R '10) each represent independently a hydrogen, an alkyl, an alkenyl or - (CH 2) m -R 8. Therefore the term "alkylamine", as used herein document, means an amine group, as defined above, which has a substituted or unsubstituted alkyl attached to it, that is, at least one of R_ {9} and R_ {10} is a group I rent.

The term "amido" is recognized in the technique as an amino substituted carbonyl e includes a remainder that can be represented by the formula general:

10

in which R_ {9}, R_ {10} are such as defined above. Preferred embodiments of the amide will not include imides that can be unstable

The term "aralkyl," as used in This document refers to an alkyl group substituted with an aryl group (for example, an aromatic group or heteroaromatic).

The term "aryl", as used in the This document includes single ring aromatic groups of 5, 6 and 7 members that can include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. The aryl groups that have heteroatoms in the ring structure can also be called "aryl heterocycles" or "heteroaromatic". The ring aromatic may be substituted at one or more ring positions with substituents such as those described above, by example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN or similar. The term "aryl" also includes systems of polycyclic ring that have two or more cyclic rings in which two or more carbons are common to two adjacent rings (the rings they are "condensed rings") in which at least one of the rings is aromatic, for example, the other cyclic rings they can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocyclyls.

The term "carbocycle", as used in This document refers to an aromatic ring or not aromatic in which each atom of the ring is carbon.

The term "carbonyl" is recognized in the technique and includes remains such as those that can be represented by the general formula:

eleven

where X is a link or represents an oxygen or a sulfur, and R 11 represents a hydrogen, an alkyl, an alkenyl, - (CH 2) m -R 8 or a salt Pharmaceutically acceptable, R '11 represents a hydrogen, a alkyl, an alkenyl or - (CH 2) m -R 8, in which m and R 8 are as defined above. When X is a oxygen and R 11 or R '11 is not hydrogen, the formula It represents an "ester." When X is an oxygen, and R 11 is as defined above, the rest is called in the present a carboxyl group, and particularly when R 11 is a hydrogen, the formula represents an "acid carboxylic ". When X is an oxygen and R '11 is hydrogen, the Formula represents a "formate." In general, when the atom of oxygen of the above formula is replaced by sulfur, the Formula represents a "thiocarbonyl" group. When X is a Sulfur and R 11 or R '11 is not hydrogen, the formula It represents a "thioester." When X is a sulfur and R_ {11} is hydrogen, the formula represents a "thiocarboxylic acid". When X is a sulfur and R '11 is hydrogen, the formula It represents a "thiolformiate." On the other hand, when X is a bond and R 11 is not hydrogen, the above formula represents a "ketone" group. When X is a link and R_ {11} is hydrogen, the above formula represents a group "aldehyde".

The term "heteroatom," as used in this document means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The terms "heterocyclyl" or "group heterocyclic "refer to ring structures from 3 to 10 members, more preferably, rings of 3 to 7 members, whose Ring structures include one to four heteroatoms. The Heterocycles can also be polycycles. The groups heterocyclyls include, for example, thiophene, thiantrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxyantiine, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, fenarsazine, phenothiazine, furazano, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams, such as azetidinones and pyrrolidinones, sultamas, sultonas, and the like. The ring heterocyclic can be substituted in one or more positions with substituents such as those described above, such as by example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, a aromatic or heteroaromatic moiety, CF3, -CN, or the like.

As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -Cl, -Br or -I; the term "sufhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO_ {2} -.

A "phosphonamidite" can be represented in The general formula:

\melm{\delm{\para}{N(R _{9} )R _{10} }}{P}{\uelm{\para}{R _{48} }}

--- O ---,

\hskip1cm

o

\hskip1cm

--- Q_{2} ---

\melm{\delm{\para}{N(R _{9} )R _{10} }}{P}{\uelm{\para}{R _{48} }}

--- OR_{46}--- Q_ {2} ---

 \ melm {\ delm {\ para} {N (R 9) R 10}} {P} {\ uelm {\ para} {R 48}}} 

--- Or ---,

 \ hskip1cm 

or

 \ hskip1cm 

--- Q_ {2} ---

 \ melm {\ delm {\ para} {N (R 9) R 10}} {P} {\ uelm {\ para} {R 48}}} 

--- OR_ {46}

in which R_ {9} and R_ {10} are as defined above, Q2 represents O, S or N, and R 48 represents a lower alkyl or an aryl, Q 2 represents O, S or N.

A "phosphoramidite" can be represented in The general formula:

\melm{\delm{\para}{N(R _{9} )R _{10} }}{P}{\uelm{\dpara}{O}}

--- O ---,

\hskip1cm

o

\hskip1cm

--- Q_{2} ---

\melm{\delm{\para}{N(R _{9} )R _{10} }}{P}{\uelm{\dpara}{O}}

--- OR_{46}--- Q_ {2} ---

 \ melm {\ delm {\ para} {N (R 9) R 10}} {P} {\ uelm {\ dpara} {O}} 

--- Or ---,

 \ hskip1cm 

or

 \ hskip1cm 

--- Q_ {2} ---

 \ melm {\ delm {\ para} {N (R 9) R 10}} {P} {\ uelm {\ dpara} {O}} 

--- OR_ {46}

in which R_ {9} and R_ {10} are as defined above and Q2 represents O, S or N.

A "phosphoryl" may be represented, in general, by the formula:

\melm{\delm{\para}{OR _{46} }}{P}{\uelm{\dpara}{Q _{1} }}

------

 \ melm {\ delm {\ para} {OR46}} {P} {\ uelm {\ dpara} {Q1}}} 

---

in which Q_ {1} represents S or O, and R 46 represents hydrogen, a lower alkyl or an aryl. When used to replace, for example, an alkyl, the group Phosphorylalkyl phosphoryl can be represented by the formula general:

\melm{\delm{\para}{OR _{46} }}{P}{\uelm{\dpara}{Q _{1} }}

--- O ---,

\hskip1cm

o

\hskip1cm

--- Q_{2} ---

\melm{\delm{\para}{OR _{46} }}{P}{\uelm{\dpara}{Q _{1} }}

--- OR_{46}--- Q_ {2} ---

 \ melm {\ delm {\ para} {OR46}} {P} {\ uelm {\ dpara} {Q1}}} 

--- Or ---,

 \ hskip1cm 

or

 \ hskip1cm 

--- Q_ {2} ---

 \ melm {\ delm {\ para} {OR46}} {P} {\ uelm {\ dpara} {Q1}}} 

--- OR_ {46}

in which Q_ {1} represents S or O, and each R 46 independently represents hydrogen, an alkyl lower or an aryl, Q2 represents O, S or N. When Q_ {1} is S, the rest of phosphoryl is a "phosphorothioate".

The terms "polycyclyl" or "group polycyclic "refers to two or more rings (for example, cycloalkyls, cycloalkenyls, cycloalkyls, aryls and / or heterocyclyls), in which two or more carbons are common to two adjacent rings, for example, the rings are "rings condensed. "The rings that are joined by atoms not Adjacent are called "bridge-forming" rings. Each of the polycycle rings may be substituted with substituents such as those described above, as per example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, a aromatic or heteroaromatic moiety, CF3, -CN, or the like.

The term "protecting group", as used herein, means temporary substituents that protect a potentially reactive functional group from unwanted chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols and acetals and ketals of aldehydes and ketones, respectively. The field of protective group chemistry has been reviewed (Greene, TW; Wuts, PGM Protective Groups in Organic Synthesis , 2nd ed .; Willey; New York, 1991).

A "selenoalkyl" refers to a group alkyl having a substituted selene group attached thereto. "Selenoethers" example that can be substituted in the group alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl and -Se- (CH 2) m-R 8, with m and R 8 being as defined above.

As used herein, the term "substituted" is considered to include all permissible substituents of organic compounds. In one aspect broad, allowable substituents include substituents acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds. Illustrative substituents include, for example, those described earlier in this document. Substituents permissible can be one or more and the same or different for appropriate organic compounds. For the purposes of this invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituent of organic compounds described herein that satisfy the valences of the heteroatoms. This invention does not intends to be limited in any way by the permissible substituents of organic compounds.

It will be understood that "substitution" or "substituted with" includes the implied condition that such substitution agrees with the allowed valence of the atom substituted and the substituent, and that the substitution results in a stable compound, for example, that does not undergo transformation spontaneously, such as by redisposition, cyclization, elimination, etc.

The term "sulfamoyl" is recognized in the technique and includes a remainder that can be represented by the General Formula:

12

in which R_ {9} and R_ {10} are as defined previously.

The term "sulfate" is recognized in the technique and includes a residue that can be represented by the formula general:

\melm{\delm{\dpara}{O}}{S}{\uelm{\dpara}{O}}

--- OR_{41}--- OR ---

 \ melm {\ delm {\ dpara} {O}} {S} {\ uelm {\ dpara} {O}} 

--- OR_ {41}

in which R_ {41} is as it is defined previously.

The term "sulfonamido" is recognized in the technique and includes a remainder that can be represented by the General Formula:

\delm{N}{\delm{\para}{R _{9} }}

---

\melm{\delm{\dpara}{O}}{S}{\uelm{\dpara}{O}}

--- R'_{11}---

 \ delm {N} {\ delm {\ para} {R9}} 

---

 \ melm {\ delm {\ dpara} {O}} {S} {\ uelm {\ dpara} {O}} 

--- R '11

in which R 9 and R '11 are as defined previously.

The term "sulfonate" is recognized in the technique and includes a residue that can be represented by the formula general:

\melm{\delm{\dpara}{O}}{S}{\uelm{\dpara}{O}}

--- OR_{41}---

 \ melm {\ delm {\ dpara} {O}} {S} {\ uelm {\ dpara} {O}} 

--- OR_ {41}

in which R_ {41} is a pair of electrons, hydrogen, alkyl, cycloalkyl or aryl

The terms "sulfoxide" or "sulfinyl", as used herein, they refer to a remainder which can be represented by the general formula:

\uelm{S}{\uelm{\dpara}{O}}

--- R_{44}---

 \ uelm {S} {\ uelm {\ dpara} {O}} 

--- R_ {44}

in which R_ {44} is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl or aryl

Similar substitutions can be made to groups alkenyl and alkynyl to produce, for example, aminoalkenyls, aminoalkyls, amidoalkenyls, amidoalkyls, iminoalkenyls, iminoalkyls, thioalkenyls, thioalkyls, alkenyls or carbonyl substituted alkyls.

As used herein, the definition of each expression, for example, alkyl, m, n, etc., when it occurs more than once in any structure, it claims be independent of its definition in another site of the same structure.

The terms triflyl, tosyl, mesyl and nonaflyl are recognized in the art and refer to trifluoromethanesulfonyl, p- toluenesulfonyl, methanesulfonyl and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate and nonaflate are recognized in the art and refer to functional groups trifluoromethanesulfonate ester, p- toluenesulfonate ester, methanesulfonate ester and nonafluorobutanesulfonate ester and to molecules containing said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p- toluenesulfonyl and methanesulfonyl, respectively. A more exhaustive list of abbreviations used by organic chemists, experts in the art, appears in the first issue of each volume of the Journal of Organic Chemistry ; This list is normally presented in a table entitled Standard List of Abbreviations . The abbreviations contained in said list and all the abbreviations used by organic chemists, experts in the art, are incorporated herein by reference.

Certain compounds of the present invention they can exist in geometric or stereoisomeric forms private individuals The present invention contemplates all those compounds, including cis and trans isomers, the R enantiomers and S, diastereomers, isomers (D), isomers (L), racemic mixtures thereof and other mixtures thereof, already that fall within the scope of the invention. May be present additional asymmetric carbon atoms in such a substituent As an alkyl group. It is intended that all such isomers as well as mixtures thereof, are included in this invention.

If desired, for example, an enantiomer particular of a compound of the present invention, may be prepared by asymmetric synthesis or by derivation with a chiral auxiliary, in which the resulting diastereomeric mixture is separates and the auxiliary group is cleaved to provide the desired pure enantiomers. Alternatively, when the molecule contains a basic functional group, such as amino, or a group functional acid, such as carboxyl, salts may be formed diastereomers with an appropriate optically active acid or base, followed by the resolution of the diastereomers thus formed by fractional crystallization or well chromatographic media known in the art, and subsequent recovery of pure enantiomers.

Compound equivalents of the compounds described above include compounds that otherwise correspond to them, and they have the same properties general ones (for example, the ability to inhibit hedgehog signaling), in which one or more variations are made simple substituents that do not adversely affect the compound efficacy In general, the compounds of the present invention can be prepared by the methods illustrated in general reaction schemes such as those described more forward, or by modifications thereof, using starting materials, reagents and synthesis procedures Conventionals readily available. In those reactions too it is possible to make use of variants that are known by themselves, but not mentioned in this document.

For the purposes of this invention, the Chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside the deck. Too For the purposes of this invention, the term is considered "hydrocarbon" includes all allowable compounds that They have at least one hydrogen and one carbon atom. In a wide aspect, allowable hydrocarbons include organic compounds acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic that may be substituted or unsubstituted.

III. Example compounds of the invention

As described in more detail below, it is considered that the object methods can be carried out using a variety of different small molecules that can be easily identified, for example, by selection tests of drugs such as those described herein. By example, the compounds useful in the subject methods include compounds that can be represented by the general formula (II):

       \ newpage
    

Formula II

13

in which, to the extent that the valence and stability what allow,

R 1 and R 2, independently for each appearance, represent H, lower alkyl, - (CH 2) n aryl (for example, substituted or not substituted), or - (CH 2) n heteroaryl (for example, substituted or unsubstituted);

L, independently for each occurrence, is absent or represents alkyl, -alkenyl-, alkynyl-, - (CH 2) n O (CH 2) p -, - (CH 2) n NR 2 (CH 2) p -, - (CH 2) n S (CH 2) p -, - (CH 2) n alkenyl (CH 2) p -, - (CH 2) n alkynyl (CH 2) p -, -O (CH 2) n -, -NR 2 (CH 2) n - or -S (CH 2) n -;

X can be selected from -N (R_ {8}) -, -O-, -S-, -Se- -N = N-, -ON = CH-, (R 8) N-N (R 8) -, -ON (R 8) -, a heterocycle, or a direct link between L e Y;

And it can be selected from -C (= O) -, -C (= S) -, -S (O2) -, -S (O) -, -C (= NCN) -, -P (= O) (OR_2) -, a heteroaromatic group, or a direct link between X and Z;

Z is selected from -N (R 8) -, -O-, -S-, -Se- -N = N-, -ON = CH-, -R_ {8} N-NR_ {8} -, -ONR 8 -, a heterocycle, or a direct link between Y and L;

R 8, independently for each occurrence, represents H, lower alkyl, - (CH2) n aryl (by example, substituted or unsubstituted), or - (CH 2) n heteroaryl (for example, substituted or not substituted), or two R 8 taken together can form a ring of 4 to 8 members, for example, with X and Z, ring that can include one or more carbonyls;

W represents a benzene or pyridonic ring, substituted or unsubstituted, condensed to the pyrimidone ring;

p represents independently for each occurrence, an integer from 0 to 10, preferably from 0 to 3; Y

n, individually for each occurrence, represents an integer from 0 to 10, preferably from 0 to 5.

In embodiments where Y_ {1} and Z_ {1} are absent and X1 comprises a pyrimidone, the compounds useful in the present invention can be represented by the formula general (II):

       \ newpage
    

Formula II

14

in which, to the extent that the valence and stability what allow,

R 1 and R 2, independently for each occurrence, represent H, lower alkyl, aryl (for example, substituted or unsubstituted), aralkyl (for example, substituted or unsubstituted, for example, - (CH 2 ) n-aryl), or heteroaryl (for example, substituted or unsubstituted), or heteroaralkyl (for example, substituted or unsubstituted, for example, - (CH2) n
heteroaralkyl);

L, independently for each occurrence, is absent or represents - (CH2) n -alkyl, -alkenyl-, alkynyl-, - (CH2) n
alkenyl-, - (CH 2) n alkynyl-, - (CH 2) n O (CH 2) p -, - (CH 2) n} NR 2 (CH 2) p -, - (CH 2) n S (CH 2) p -, - (CH 2) _ {n} alkenyl (CH2) p -, - (CH2) n
alkynyl (CH 2) p -, -O (CH 2) n -, -NR 2 (CH 2) n - or -S (CH 2 ) n -; which may be optionally substituted by a group selected from H, substituted or unsubstituted lower alkyl, alkenyl or alkynyl, cycloalkylalkyl (for example, substituted or unsubstituted, for example, - - (CH2) n cycloalkyl), (for example, substituted or unsubstituted), aryl (for example, substituted or unsubstituted), aralkyl (for example, substituted or unsubstituted, for example, - (CH2) n aryl), or heteroaryl (for example, substituted or unsubstituted), or heteroaralkyl (for example, substituted or unsubstituted, for example,
- (CH 2) n heteroaralkyl), preferably H, lower alkyl, - (CH 2) n aryl (for example, substituted or unsubstituted), or - (CH 2 ) n heteroaryl (for example, substituted or unsubstituted);

X can be selected from -N (R_ {8}) -, -O-, -S-, -Se- -N = N-, -ON = CH-, - (R 8) N-N (R 8) -, -ON (R 8) -, a heterocycle, or a direct link between L e Y;

And it can be selected from -C (= O) -, -C (= S) -, -S (O2) -, -S (O) -, -C (= NCN) -, -P (= O) (OR_2) -, a heteroaromatic group, or a direct link between X and Z;

Z is selected from -N (R 8) -, -O-, -S-, -Se- -N = N-, -ON = CH-, -R_ {8} N-NR_ {8} -, -ONR 8 -, a heterocycle, or a direct link between Y and L;

R 8, independently for each occurrence, represents H, lower alkyl, aryl (for example, substituted or not substituted), aralkyl (for example, substituted or unsubstituted, by example, - (CH 2) n aryl), or heteroaryl (for example, substituted or unsubstituted), or heteroaralkyl (for example, substituted or unsubstituted, for example, - (CH 2) n heteroaralkyl, or two R 8 taken together they can form a ring of 4 to 8 members, for example, with X and Z, ring that may include one or more carbonyls;

W represents a benzene or pyridonic ring, substituted or unsubstituted, condensed to the pyrimidone ring;

p represents independently for each occurrence, an integer from 0 to 10, preferably from 0 to 3; Y

n, individually for each occurrence, represents an integer from 0 to 10, preferably from 0 to 5.

In certain embodiments, R_ {1} represents a substituted or unsubstituted aryl or heteroaryl group, for example, a phenyl ring, a pyridine ring, etc. In certain embodiments in which -LR_ {1} represents an aryl group or substituted heteroaryl, R 1 is preferably not substituted with an isopropoxyl group (Me2CHO-). In certain embodiments in which -LR1 represents an aryl or heteroaryl group substituted, R1 is preferably not substituted with a group ether. In certain embodiments, the substituents in R1 (for example, other than hydrogen) are selected from halogen, cyano, alkyl, alkenyl, alkynyl, aryl, hydroxyl, (unbranched alkyl-O-), silyloxy, amino, nitro, thiol, amino, imino, amido, phosphoryl, phosphonate, phosphine, carbonyl, carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl, arylsulfonyl, sulfoxide, selenoether, ketone, aldehyde, ester or - (CH 2) m -R 8. In certain embodiments, substituents other than hydrogen are select from halogen, cyano, alkyl, alkenyl, alkynyl, aryl, nitro, thiol, imino, amido, carbonyl, carboxyl, anhydride, thioether, alkylsulfonyl, arylsulfonyl, ketone, aldehyde and ester. In certain embodiments, substituents other than hydrogen are selected from halogen, cyano, alkyl, alkenyl, alkynyl, nitro, amido, carboxyl, anhydride, alkylsulfonyl, ketone, aldehyde and ester.

In certain embodiments, X can be selected of -N (R_ {8}) -, -O-, -S-, a direct link, and a heterocycle, Y can be selected from -C (= O) -, -C (= S) - and -S (O_ {2}) -, and Z can be selected from -N (R_ {8}) -, -O-, -S-, a direct link and a heterocycle. In certain such embodiments, at least one of Z and X is present.

In certain related embodiments, X-Y-Z taken together represent a urea (NC (O) N) or an amide (NC (O) or WITH).

In certain embodiments, W is a ring of substituted or unsubstituted benzene.

In certain embodiments, X represents a diazacarbocycle, such as a piperazine, for example, substituted or not replaced

In certain embodiments, X can be selected of -N (R_ {8}) -, -O-, -S- and a direct link, and can be selected from -C (= O) -, -C (= S) - and -S (O_ {2}) -, and Z can be selected from -N (R_ {8}) -, -O-, -S- and a direct link, so that at least one of X and Z is present.

In certain embodiments, R 8 represents H, lower alkyl, aralkyl, heteroaralkyl, aryl or heteroaryl, for example, H or lower alkyl.

In certain embodiments, X represents -NH-.

In certain embodiments, -L-X- represents - (lower unbranched alkyl) -NH-, for example, -CH2 -NH-, -CH_ {2} -CH_ {2} -NH-, etc.

In certain embodiments, the object antagonists may be chosen based on their selectivity for the hedgehog route. This selectivity can be for the hedgehog route versus other routes, or for the selectivity between particular hedgehog routes, for example, ptc-1, ptc-2 , etc.

In certain preferred embodiments, the subject inhibitors inhibit hedgehog- mediated signal transduction with an ED50 of 1 mM or less, more preferably 1 µM or less, and even more preferably 1 nM or less.

In particular embodiments, the small molecule is chosen for use because it is more selective for a patched isoform with respect to the following, for example, 10 times and, more preferably, at least 100 or even 1000 times more selective for a patched path ( ptc-1, ptc-2 ) with respect to another.

In certain embodiments, a compound that is an antagonist of the hedgehog pathway is chosen to selectively antagonize the activity of hedgehog with respect to protein kinases other than PKA, such as PKC, for example, the compound modulates the activity of the hedgehog pathway. less an order of magnitude more strongly that modulates the activity of another protein kinase, preferably at least two orders of magnitude more strongly, even more preferably at least three orders of magnitude more strongly. For example, a preferred pathway inhibitor hedgehog can inhibit the activity of hedgehog with a K_ {i} at the least an order of magnitude lower than its K {i} for inhibition of PKC, preferably at the least two orders of lower magnitude, even more preferably at least three orders of lower magnitude. In certain embodiments, the Ki for PKA inhibition is less than 10 nM, preferably less than 1 nM, even more preferably less than 0.1 nM.

Synthesis of the subject compounds

In certain embodiments, the present invention refers to techniques and approaches for the synthesis of compounds object, as described by the formulas I-VIII.

For example, in certain embodiments, the The present invention relates to a method for preparing a compound object as explained in scheme I,

       \ newpage
    

Scheme I

fifteen

in which stage (A) comprises reacting a compound having a structure of formula X, wherein W represents an aryl or heteroaryl ring, substituted or unsubstituted, such as a benzene ring, which has a group amino and a group of carboxylic acid in adjacent positions (ortho), with an acylation agent having the formula R 10 CH 2 C (= O) X ', in which R 10, regardless of each occurrence, it represents alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or heteroaralkyl, substituted or unsubstituted, and X 'represents a halogen or -OC (= O) CH 2 R 10, under conditions that produce a compound that has a formula structure XI;

step (B) comprises reacting a compound having a structure of formula XI with an amine that it has the formula R 1 LNH 2, in which R 1 and L are such as defined above, under conditions that give as result a compound having a structure of formula XII;

step (C) comprises reacting a compound having a structure of formula XII with an agent halogenating, such as chlorine, bromine, iodine, N-Bromosuccinimide, N-Chlorosuccinimide, N-iodosuccinimide, ClBr, IBr, ClI, or a reagent which generates a halogen radical (such as Cl; Br; or I) under conditions which result in a compound that has a structure of formula XIII, in which Y 'represents a halogen such as Cl, Br or I;

step (D) comprises reacting a compound having a structure of formula XIII with an amine that it has the formula H2 {NR_ {12}, in which R_ {12} represents a lower alkyl group or a silyl group, such as a group trialkylsilyl, triarylsilyl, dialkylarylsilyl or diarylalkylsilyl, under conditions that result in a compound having the structure of formula XIV; Y

step (E) comprises reacting a compound having a structure of formula XIV with a termination group having a structure of R2 V 'to produce a compound having a structure of formula XX, in which R_ { 2} is as defined above and V 'represents a functional group selected from ZC (= W) Cl, ZC (= W) Br, isocyanate, isothiocyanate, ZC (= W) WC (= W) ZR 2, ZSO 2 Cl, ZSO 2 Br, ZSOCl, ZSOBr, or an activated acylation moiety prepared in situ .

In certain embodiments, an object compound can be prepared by providing a compound that has a structure of formula XI and performing steps (B) to (E). In certain embodiments, an object compound can be prepared providing a compound having a structure of formula XII and performing steps (C) to (E). In certain embodiments, a object compound can be prepared by providing a compound that It has a structure of formula XIII and performing steps (D) to (AND). In certain embodiments, an object compound may be prepared. providing a compound having a structure of formula XIV and performing stage (E).

In certain embodiments, step (A) may carried out using an anhydride, such as an anhydride symmetric, which has the formula R 10 C (= O) OC (-O) R 10. In certain embodiments, The reaction can be carried out using the anhydride as a solvent, for example, the reaction mixture is substantially free of a solvent other than anhydride. In certain embodiments, R 10, for both occurrences, is an alkyl group lower.

In certain embodiments, step (B) may be carried out by treating a compound of formula XI with an amine in a non-polar solvent, such as chloroform, methylene chloride, ethylene chloride, toluene, benzene, ether, tetrahydrofuran, or other non-polar solvent, or any combination thereof, followed for treating the product of that reaction with an ionic base, such as an alkali metal alkoxide or hydroxide, such as methoxide, ethoxide, etc., in a polar solvent, such as a solvent alcoholic, for example, methanol, ethanol, propanol, ethylene glycol, propylene glycol, glycerol, etc. In certain embodiments, the conditions may include heating the reaction mixture above from room temperature, for example, to at least 50 ° C, or at minus 75 ° C, or even at least 100 ° C. In certain embodiments, this step can convert a compound of formula X into a compound of formula XI in at least about 80% yield, or at less about 85% yield.

In certain embodiments, step (C) may carried out using a halogen as a halogenating agent, and it can be carried out in a reaction mixture that includes an acid carboxylic, such as acetic acid or propionic acid. In certain embodiments, may be added or present in the mixture of reaction, a soft base, such as a carboxylic acid salt.

In certain embodiments, step (D) may carried out using a polar solvent, such as water, ethanol, methanol, ethylene glycol, DMF, or other polar solvent, or any combination of polar solvents. In certain embodiments, the solvent or solvent mixture comprises less of approximately 50% water, or it is non-aqueous, that is, it is substantially free of water. In certain embodiments, the Polar solvent comprises an alcohol, such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butanol, sec-butanol, ethylene glycol  and 1,3-propanediol.

In certain embodiments, step (E) can be carried out by generating an acylation agent in situ , for example, by reacting a carboxylic acid with an activating agent, such as a carbodiimide (for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1- ( 3-dimethylaminopropyl) -3-ethylcarbodiimide, etc.), phosphorus-based reagents (such as BOP-Cl, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, or any other reagent that reacts with a carboxylic acid group , resulting in a reactive intermediate that has an increased sensitivity, with respect to carboxylic acid, towards coupling with an amine. A wide variety of such reagents are well known in the art of organic synthesis, especially peptide coupling. Similarly, a primary amine can be treated with a phosgene equivalent, such as carbonyldiimidazole, phosgene, triphosgene, diphosgene, etc., or a thiophosgene equivalent, such as thiophosgene, thiocarbonyldiimidazole, etc., to generate an acylating agent ( for example, an isocyanate, isothiocyanate, chloroformamide or chlorothioformamide, for example) capable of reacting with an amine to form a urea or thiourea, without requiring isolation or purification of the acylating agent.

In certain embodiments, the present invention refers to a method for preparing an object compound as it is explained in scheme II,

       \ vskip1.000000 \ baselineskip
    

16

in which stage (A) comprises reacting a compound that has a formula structure XV, in which P 'represents H or a protecting group, W represents a aryl or heteroaryl ring, substituted or unsubstituted, such as a benzene ring, which has an amino group and an acid group carboxylic or an ester group in adjacent positions (ortho), with a acylating agent having the formula PXLC (= O) X ', in the that X and L are as defined above, P represents a protective group and X 'represents a halogen -OC (= O) LXP, or a functional group generated by reacting a carboxyl group with an activating agent, such as a carbodiimide (for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, etc.), phosphorus-based reagents (such as BOP-Cl, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, carbonyldiimidazole or any other reagent that react with a carboxylic acid group, resulting in a reactive intermediate that has an increase in sensitivity, with respect to carboxylic acid, towards coupling with an amine, under conditions that produce a compound which has a formula structure XVI;

step (B) comprises deprotecting the ester of a compound having a structure of formula XVI to produce a carboxylic acid having a structure of formula XVII, if it is necessary;

step (C) comprises reacting a compound having a structure of formula XVII with an amine that it has the formula R 1 LNH 2, in which R 1 and L are such as defined above, under conditions that give as result a compound that has a formula structure XVIII;

step (D) comprises removing the group P protector of a compound having a formula structure XVIII to generate a compound that has a formula structure XIX;

step (E) comprises reacting a compound having a structure of formula XIX with a termination group having a structure of R 2 Y 'to produce a compound having a structure of formula XXI, in which R_ { 2} is as defined above, and Y 'represents a functional group selected from ZC (= W) Cl, ZC (= W) Br, isocyanate, isothiocyanate, ZC (= W) WC (= W) ZR2} , ZSO 2 Cl, ZSO 2 Br, ZSOCl, ZSOBr, or an active acylation moiety prepared in situ .

In certain embodiments, an object compound can be prepared by providing a compound that has a structure of formula XVI and performing steps (B) to (E). In certain embodiments, an object compound can be prepared providing a compound having a structure of formula XVII and performing steps (C) to (E). In certain embodiments, a object compound can be prepared by providing a compound that It has a structure of formula XVIII and performing the steps (D) to (AND). In certain embodiments, an object compound may be prepared. providing a compound having a structure of formula XIX and performing stage (E).

In certain embodiments, step (B) may be carried out by treating a compound of formula XVI with an acid in presence of water, or with a hydroxide base (for example, for hydrolyze or saponify an ester), by hydrogenolysis (by example, to remove benzyl or allyl esters), in presence of a soft base (for example, to remove an ester fluorenylmethyl), adding a Lewis acid (for example, to remove a methoxymethyl ester), in the presence of a fluoride ion (for example, to remove an ester 2-silylethyl), or any other means suitable.

In certain embodiments, step (C) may carried out by activating the carboxyl group of the compound of formula XVII with an activating agent, such as a carbodiimide (for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, etc.), phosphorus-based reagents (such as BOP-Cl, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, carbonyldiimidazole, or any other reagent that react with a carboxylic acid group, resulting in a reactive intermediate that has an increase in sensitivity, with respect to carboxylic acid, towards coupling with an amine. A wide variety of such reagents they are well known in the art of organic synthesis, especially the peptide coupling. In certain embodiments, the conditions they may include heating the reaction mixture above the room temperature, for example, up to at least 50 ° C, or at least 75 ° C, or even at least 100 ° C.

In certain embodiments, step (D) may carried out by hydrogenolysis (for example, to eliminate an Alloc or CBz group), using a base or a hydride reagent (for example, to remove a trifluoroacetyl group), using an acid, such as trifluoroacetic acid (for example, for remove a BOC group), or by any other suitable means to protect the amine.

In certain embodiments, step (E) can be carried out by generating an acylation agent in situ , for example, by reacting a carboxylic acid with an activating agent, such as a carbodiimide (for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1- ( 3-dimethylaminopropyl) -3-ethylcarbodiimide, etc.), phosphorus-based reagents (such as BOP-Cl, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, or any other reagent that reacts with a carboxylic acid group , resulting in a reactive intermediate that has an increased sensitivity, with respect to carboxylic acid, towards coupling with an amine. A wide variety of such reagents are well known in the art of organic synthesis, especially peptide coupling. Similarly, a primary amine can be treated with a phosgene equivalent, such as carbonyldiimidazole, phosgene, triphosgene, diphosgene, etc., or a thiophosgene equivalent, such as thiophosgene, thiocarbonyldiimidazole, etc. to generate an acylating agent (for example, an isocyanate, isothiocyanate, chloroformamide or chlorothioformamide, for example) capable of reacting with an amine to form a urea or thiourea, without requiring isolation or purification of the acylating agent.

IV. Example applications of the method and compositions

Another aspect of the present invention relates to to a method of modulating a differentiated state, survival and / or the proliferation of a cell that has a gain phenotype of hedgehog function, contacting cells with a hedgehog antagonist, according to the object method and as they can Justify the circumstances.

For example, the invention contemplates that, in light of the findings of a seemingly broad participation of hedgehog, ptc and smoothened in the formation of ordered spatial arrangements of differentiated tissues in vertebrates, the object method could be used as part of a procedure to generate and / or maintain a series of different vertebrate tissues both in vitro and in vivo . The hedgehog antagonist, whether inductive or anti-inductive with respect to the proliferation or differentiation of a given tissue, may, as appropriate, be any of the preparations described above.

For example, the present method can be applied to cell culture techniques, in which, whether for genetic or biochemical reasons, the cells have a hedgehog function gain phenotype. In vitro , neuronal culture systems have proven to be fundamental and indispensable tools for the study of neuronal development, as well as for the identification of neurotrophic factors, such as nerve growth factor (NGF), trophic ciliary factors (CNTF), and brain-derived neurotrophic factor (BDNF). A use of the present method may be in cultures of neuronal stem cells, such as in the use of such cultures for the generation of new neurons and glia. In such embodiments of the object method, cultured cells may be contacted with a hedgehog antagonist of the present invention, in order to modify the proliferation rate of neuronal stem cells in the culture and / or modify the differentiation rate, or to maintain the integrity of a culture of certain terminally differentiated neuronal cells. In an example embodiment, the object method can be used for the culture, for example, of sensory neurons or, alternatively, motor neurons. Such neuronal cultures can be used as convenient assay systems, as well as implantable cell sources for therapeutic treatments.

According to the present invention, large numbers of non-tumorigenic neuronal progenitor cells can be perpetuated in vitro and their proliferation and / or differentiation rate can be affected by contact with hedgehog antagonists of the present invention. In general, a method is provided comprising the steps of isolating the neuronal progenitor cells of an animal, perpetuating these cells in vitro or in vivo , preferably in the presence of growth factors and regulating the differentiation of these cells into particular neuronal phenotypes, by example, neurons and glia, contacting the cells with a hedgehog antagonist.

It is believed that the progenitor cells are under a tonic inhibitory influence that keeps parents in an inhibited state until differentiation is required. Without However, recent techniques have been provided that allow these cells proliferate and, unlike the neurons that are terminally differentiated and therefore do not divide, can occur in an unlimited number and are extremely suitable for the transplant in heterologous and autologous hosts with diseases neurodegenerative

By "parent" is meant a cell oligopotent or multipotent mother that can be divided without limit and, under specific conditions, it can produce daughter cells that they differ terminally, such as in neurons and glia. These cells can be used for transplantation in a host heterologous or autologous. A heterologist means a host other than the animal from which the cells originally derived parents. Autologous means the identical host of the that originally derived cells.

Cells can be obtained from neuronal tissue embryonic, postnatal, infant or adult. Through any animal it means any multicellular animal that contains tissue nervous. More particularly, any fish is meant, reptile, bird, amphibian or mammal and the like. Donors more Preferred are mammals, especially mice and beings humans.

In the case of a heterologous donor animal, the animal can be sacrificed and the brain and area removed specific interest using a sterile procedure. The areas of the brain of particular interest include any zone of which can be obtained progenitor cells, which will serve to restore function to a degenerated area of the host's brain. These regions include areas of the central nervous system (CNS), including the cerebral cortex, cerebellum, midbrain, trunk brain, spinal cord and ventricular tissue and system areas peripheral nervous (SNP) including the carotid body and the adrenal medulla More particularly, these areas include regions in the basal ganglia, preferably in the body striated consisting of the caudate and putamen, or several groups of cells, such as the pale globe, the subthalamic nucleus, the nucleus baseline found to be degenerated in patients with Alzheimer's disease, or the compact black substance that is finds that it is degenerated in patients with Parkinson's

Heterologous neuronal progenitor cells human can be derived from fetal tissue obtained from abortion provoked, or from a donor of postnatal, infant or adult organs. Autologous neuronal tissue can be obtained by biopsy, or from patients undergoing neurosurgery in which tissue is removed neuronal, particularly during epilepsy surgery and, more particularly during lobectomies and excisions of the Temporary hippocampus

Cells can be obtained from donor tissue by decoupling individual matrix cells extracellular tissue connection. Dissociation can be obtained using any known procedure, including treatment with enzymes, such as trypsin, collagenase and the like, or using physical methods of dissociation, such as with a blunt instrument or itching with a scalpel to allow external growth of the specific cell types of a tissue. The dissociation of fetal cells can be carried out in a culture medium tissue, while a preferred means for the dissociation of Infant and adult cells is the cerebrospinal fluid artificial (CSF). Regular CSF contains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl 2, 2 mM CaCl 2, 26 mM NaHCO 3 and 10 mM G-glucose. The low CSF in Ca 2+ contains the same components, except that MgCl2 has a 3.2 mM concentration and CaCl2 has a concentration of 0.1 mM.

Dissociated cells can be placed in any known culture medium that can withstand the cell growth, including MEM, DMEM, RPMI, F-12 and similar, which contain contributions supplements that are required for cell metabolism, such like glutamine and other amino acids, vitamins, minerals and proteins useful such as transferrin and the like. The medium can also contain antibiotics to prevent contamination with yeasts, bacteria and fungi, such as penicillin, streptomycin, gentamicin and the like. In some cases, the medium may contain serum derived from bovine, equine, chicken and the like. A medium particularly preferred for cells is a mixture of DMEM and F-12

The conditions for cultivation must be close to physiological conditions. The pH of the media of culture should be close to physiological pH, preferably between pH 6-8, more preferably close to pH 7, even more particularly about pH 7.4. Cells must grown at a temperature close to the physiological temperature, preferably between 30 ° C-40 ° C, more preferably between 32ºC-38ºC and more preferably between 35 ° C-37 ° C.

The cells can be grown in suspension or on a fixed substrate, but the proliferation of the parents is preferably performed in suspension to generate large numbers of cells by forming "neurospheres" (see, for example, Reynolds et al (1992) Science 255: 1070-1709; PCT publications WO93 / 01275, WO94 / 09119, WO94 / 10292 and WO94 / 16718). In the case of propagation (or division) of the suspended cells, the flasks are well shaken and the neurospheres are allowed to settle in a corner of the bottom of the flask. The spheres are then transferred to a 50 ml centrifuge tube and centrifuged at low speed. The medium is aspirated, the cells are resuspended in a small amount of medium with growth factor, and the cells are mechanically dissociated and resuspended in aliquots separated from the media.

Cell suspensions in the culture medium they complement each other with any growth factor that allows proliferation of progenitor cells and are seeded in any container that can hold the cells, although such as explained above, preferably in flasks of culture or rotating bottles. The cells normally proliferate in a period of 3-4 days in an incubator at 37 ° C, and the proliferation can be restarted at any time after this by dissociating the cells and resuspension in between recent that contains growth factors.

In the absence of the substrate, the cells detach from the bottom of the flask and continue to proliferate in the suspension forming a hollow sphere of undifferentiated cells. After approximately 3-10 days in vitro , proliferation clusters (neurospheres) are fed every 2-7 days, and more particularly every 2-4 days by gentle centrifugation and resuspension in medium containing growth factor.

After 6-7 days in vitro , the individual cells in the neurospheres can be separated by physical dissociation of the neurospheres with a blunt instrument, more particularly by crushing the neurospheres with a pipette. The individual cells from the dissociated neurospheres are suspended in culture medium containing growth factors, and the differentiation of the cells can be controlled in the culture by plating (or resuspend) the cells in the presence of a hedgehog antagonist.

To further illustrate other uses of the object hedgehog antagonists, it is noted that intracerebral grafting has emerged as an additional approach for central nervous system treatments. For example, an approach to repair injured brain tissues involves the transplantation of fetal or neonatal animal cells in the adult's brain (Dunnett et al . (1987) J Exp Biol 123: 265-289; and Freund et al . (1985) J Neurosci 5: 603-616). Fetal neurons from a variety of brain regions can be successfully incorporated into the brain of the adult, and such grafts can alleviate behavioral defects. For example, movement disorder induced by lesions of dopaminergic projections to the basal ganglia can be avoided by grafts of embryonic dopaminergic neurons. Complex cognitive functions that are altered after neocortex lesions can also be partially restored by grafts of embryonic cortical cells. The object method can be used to regulate the growth state in the culture, or when fetal tissue is used, especially neuronal stem cells, it can be used to regulate the rate of differentiation of the cells.
mother.

Stem cells useful in the present invention are generally known. For example, several cells of the neural crest have been identified, some of which are multipotent and probably represent non-compromised neural crest cells, and others can generate only one type of cell, such as sensory neurons, and probably represent cells committed parents. The role of hedgehog antagonists employed in the present method for culturing such stem cells may be to regulate the differentiation of the non-compromised parent, or to regulate the additional restriction of fate in the development of a compromised progenitor cell towards becoming a terminal differentiated neuronal cell . For example, the present method can be used in vitro to regulate the differentiation of neural crest cells into neurogliocytes, Schwann cells, chromaffin cells, parasympathetic or sympathetic cholinergic neurons, as well as peptidergic or serotonergic neurons. Hedgehog antagonists can be used alone, or they can be used in combination with other neurotrophic factors that act to more particularly intensify a particular differentiation destination of the progenitor cell.
neuronal

In addition to the implantation of cultured cells in the presence of object hedgehog antagonists, yet another aspect of the present invention relates to the therapeutic application of a hedgehog antagonist to regulate the growth status of neurons and other neuronal cells, both in the central nervous system as in the peripheral nervous system. The ability of ptc, hedgehog and smoothened to regulate neuronal differentiation during the development of the nervous system and also presumably in the adult state, indicates that, in certain cases, the object hedgehog antagonists can be expected to facilitate the control of adult neurons with regarding the maintenance, functional performance and aging of normal cells; repair and regeneration processes in chemically or mechanically injured cells; and the treatment of degeneration in certain pathological states. In the light of this understanding, the present invention specifically contemplates applications of the object method for the treatment protocol of (prevention and / or reduction of the severity of) neurological states that derive from: (i) acute, subacute or chronic injury to the central nervous system, including trauma injury, chemical injury, vascular injury and deficiencies (such as ischemia resulting from stroke), along with infectious / inflammatory and tumor-induced injury; (ii) aging of the nervous system, including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations; and (iv) chronic immune diseases of the nervous system or that affect the nervous system, including multiple sclerosis.

As appropriate, the object method can also be used in the generation of nerve prostheses for the repair of the lesion of the central and peripheral nerve. In particular, when a crushed or cut axon is intubated through the use of a prosthetic device, hedgehog antagonists can be added to the prosthetic device to regulate the growth rate and regeneration of dendritic processes. Example nerve guide channels are described in US Pat. Nos. 5,092,871 and 4,955,892.

In another embodiment, the object method can be used in the treatment of neoplastic or hyperplastic transformations, as can occur in the central nervous system. For example, hedgehog antagonists can be used to produce transformed cells such that they become posmitotic or apoptotic. Therefore, the present method can be used as part of a treatment for, for example, malignant gliomas, meningiomas, medulloblastomas, neuroectodermal tumors and ependymomas.

In a preferred embodiment, the object method can be used as part of a treatment regimen for malignant medulloblastoma and other primitive neuroectodermal tumors CNS malignant.

In certain embodiments, the object method is used as part of the treatment program for medulloblastoma. The medulloblastoma, a primary brain tumor, is the brain tumor More common in children. A medulloblastoma is a neuroectodermal tumor primitive that arises in the posterior fossa. Constitute approximately 25% of all childhood brain tumors (Miller) Histologically, they are small cell tumors and round commonly arranged in true rosettes, but may manifest some differentiation to astrosites, ependymocytes or neurons (Rorke; Kleihues). PNET may arise (tumors primitive neuroectodermal) in other areas of the brain, including the pineal gland (pineoblastoma) and the brain. Those who arise in the supratentorial region, usually evolve worse than their PF counterparts (from the posterior fossa).

It is known that medulloblastoma / PENT recurs anywhere in the CNS after resection and can become metastatic to the bone. Therefore, the pre-treatment evaluation should include an examination of the spinal cord to exclude the possibility of masses (" dropped metastases "). MRI (magnetic resonance imaging) enhanced by gadolinium has largely replaced myelography for this purpose, and CSF cytology is obtained postoperatively as a routine procedure.

In other embodiments, the object method is used. as part of the treatment program for ependymomas. The Ependymomas constitute approximately 10% of tumors brain in children. Roughly speaking, they are tumors that arise from ependymal lining of the ventricles and that microscopically they form rosettes, channels and perivascular rosettes. In the series of CHOP of 51 children who had been reported to have ependymomas, 3/4 They were histologically benign. Approximately 2/3 arose from the region of the 4th ventricle. A third showed up in the region supratentorial The age at the presentation peaks is between the birth and 4 years, as evidenced by data from SEER, as well as the CHOP data. The average age is approximately 5 years. Because many children with this disease are babies, often require multimodal treatment.

Yet another aspect of the present invention relates to the observation in the art that ptc, hedgehog and / or smoothened participate in the morphogenic signals involved in other organogenic vertebrate pathways, in addition to neuronal differentiation, as described above, having obvious roles in other formations of endodermal pattern, as well as in the processes of mesodermal and endodermal differentiation. Therefore, the invention contemplates that compositions comprising hedgehog antagonists can also be used for both cell culture and therapeutic methods that involve the generation and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery that ptc, hedgehog and smoothened apparently participate in controlling the development of the stem cells responsible for the formation of the digestive tract, liver, lungs and other organs that are derived from the primitive intestine. Shh serves as an inducing signal from the endoderm to the mesoderm, which is critical for bowel morphogenesis. Thus, for example, the hedgehog antagonists of the present method can be used to regulate the development and maintenance of an artificial liver that can have multiple metabolic functions of a normal liver. In an example embodiment, the object method can be used to regulate the proliferation and differentiation of the stem cells of the digestive tract to form hepatocyte cultures that can be used to populate extracellular matrices, or that can be encapsulated in biocompatible polymers, to form implantable artificial livers. and extracorporeal.

In another embodiment, the therapeutic compositions of hedgehog antagonists can be used in conjunction with the transplantation of such artificial livers, as well as embryonic liver structures, to regulate intraperitoneal implant uptake, vascularization and differentiation and in vivo maintenance of liver tissue. grafted

In yet another embodiment, the object method can be used therapeutically to regulate such organs after physical, chemical or pathological attack. For example, therapeutic compositions comprising hedgehog antagonists can be used in the repair of the liver after a partial hepatectomy.

The generation of the pancreas and small intestine from the embryonic intestine depends on the intercellular signaling between the endodermal and mesodermal cells of the intestine. In particular, it has been suggested that differentiation of the intestinal mesoderm in the smooth muscle depends on signals from adjacent endodermal cells. A candidate mediator of signals derived from the endoderm in the posterior embryonic gut is Sonic hedgehog. See, for example, Apelqvist et al . (1997) Curr Biol 7: 801-4. The Shh gene is expressed throughout the embryonic gut endoderm, with the exception of the pancreatic bud endoderm, which instead expresses high levels of the homeodomain protein lpf1 / Pdx1 (insulin promoting factor 1 / pancreatic and duodenal homeotic box 1), an essential regulator of early pancreatic development. Apelqvist et al ., Cited above, have examined whether the differential expression of Shh in the embryonic gut tube controls the differentiation of the surrounding mesoderm into specialized derivatives of the small intestine mesoderm and pancreas. To prove this, they used the lpf1 / Pdx1 gene promoter to selectively express Shh in the development of the pancreatic epithelium. In the transgenic mouse lpf1 / Pdx1 - Shh, the pancreatic mesoderm develops in smooth muscle and interstitial cells of Cajal, characteristic of the intestine, rather than in pancreatic mesenchyme and spleen. In addition, pancreatic explants exposed to Shh underwent a similar program of intestinal differentiation. These results show that the differential expression of endogenously derived Shh controls the fate of the adjacent mesoderm in different regions of the intestinal tube.

In the context of the present invention, it is therefore contemplated that the object hedgehog antagonists can be used to control or regulate the proliferation and / or differentiation of pancreatic tissue both in vivo and in vitro .

There is a wide variety of pathological states of cell proliferation and differentiation for which the inhibitors of the present invention can provide therapeutic benefits, the general strategy being, for example, the correction of aberrant insulin expression, or the modulation of differentiation. . However, more generally, the present invention relates to a method of inducing and / or maintaining a differentiated state, enhancing survival and / or affecting the proliferation of pancreatic cells, by contacting the cells with the object inhibitors. For example, the invention contemplates that, in light of the apparent involvement of ptc, hedgehog and smoothened in the formation of ordered spatial arrangements of pancreatic tissues, the object method could be used as part of a technique to generate and / or maintain such tissue, both in vitro and in vivo . For example, hedgehog function modulation can be used both in cell culture and in therapeutic methods that involve the generation and maintenance of β cells and also possibly for non-pancreatic tissue, such as in the control of the development and maintenance of tissue from the digestive tract, spleen, lungs, urogenital organs (for example, the bladder) and other organs that are derived from the primitive intestine.

In an exemplary embodiment, the present method can be used in the treatment of hyperplastic and neoplastic disorders that affect pancreatic tissue, particularly those characterized by aberrant proliferation of pancreatic cells. For example, pancreatic cancers are characterized by abnormal proliferation of pancreatic cells that can result in alterations in the secretion capacity of the pancreas insulin. For example, certain pancreatic hyperplasias, such as pancreatic carcinomas, may result in hypoinsulinemia due to β cell dysfunction or decreased islet cell mass. To the extent that aberrant signaling of ptc, hedgehog and smoothened can be indicated in the progression of the disease, the object inhibitors can be used to enhance tissue regeneration after anti-tumor treatment.

In addition, manipulation of hedgehog signaling properties at different points may be useful as part of a pancreatic tissue restructuring / repair strategy both in vivo and in vitro . In one embodiment, the present invention makes use of the apparent involvement of ptc, hedgehog and smoothened in the regulation of pancreatic tissue development. In general, the object method can be used therapeutically to regulate the pancreas after physical, chemical or pathological attack. In yet another embodiment, the object method can be applied to cell culture techniques and, in particular, can be used to enhance the initial generation of prosthetic devices of pancreatic tissue. Manipulation of proliferation and differentiation of pancreatic tissue, for example, by altering hedgehog activity, can provide a means to more carefully control the characteristics of a cultured tissue. In an example embodiment, the object method can be used to increase the production of prosthetic devices that require islets of β cells, such as in the encapsulation devices described, for example, in US Pat. granted to Aebischer et al . No. 4,892,538, U.S. Pat. granted to Aebischer et al . No. 5,106,627, U.S. Pat. granted to Lim number 4,391,909 and US Pat. granted to Sefton number 4,353,888. The initial progenitor cells for pancreatic islets are multipotential and apparently coactivate all the specific genes of the islet from the moment they first appear. As development progresses, the expression of islet-specific hormones, such as insulin, is restricted to the characteristic expression pattern of mature islet cells. However, the phenotype of mature islet cells is not stable in culture, since the reappearance of embryonic traits in mature? Cells can be observed. By using the object hedgehog antagonists, the differentiation path or proliferative index of the cells can be regulated.

In addition, manipulation of the state of differentiation of pancreatic tissue can be used in conjunction with artificial pancreas transplantation, so as to improve implantation, vascularization and differentiation and in vivo maintenance of grafted tissue. For example, manipulation of the hedgehog function can be used to affect tissue differentiation as a means to maintain the viability of the graft.

Bellusci et al . (1997) Development 124: 53 report that Sonic hedgehog regulates the proliferation of lung mesenchymal cells in vivo . Consequently, the present method can be used to regulate the regeneration of lung tissue, for example, in the treatment of emphysema.

Fujita et al . (1997) Biochem Biophys Res Commun 238: 658 reported that Sonic hedgehog is expressed in adenocarcinoma cells and squamous lung carcinoma in humans. Sonic hedgehog expression was also detected in lung squamous carcinoma tissues in humans, but not in normal lung tissue of the same patient. They also observed that Sonic hedgehog stimulates the incorporation of BrdU into carcinoma cells and stimulates their cell growth, while anti-Shh-N inhibited their cell growth. These results suggest that ptc, hedgehog and / or smoothened are involved in the cellular growth of such transformed lung tissue and, therefore, indicates that the subject method can be used as part of a treatment of pulmonary adenocarcinomas and carcinoma, and other proliferative disorders that They involve the pulmonary epithelium.

Based on evidence such as the involvement of the hedgehog pathway in these tumors, or the detected expression of hedgehog or its receptor in these tissues during development, many other tumors may be affected by treatment with the subject compounds. Such tumors include, but are by no means limited to, tumors related to Gorlin syndrome (eg, medulloblastoma, meningioma, etc.), tumors demonstrated in deficient mice (knock-out) in pct (eg, hemangioma, rhabdomyosarcoma , etc.), tumors resulting from the amplification of gli-1 (for example, glioblastoma, sarcoma, etc.), TRC8-related tumors, a homologue of ptc (for example, renal carcinoma, thyroid carcinoma, etc.), Ext-1 related tumors (for example, bone cancer, etc.), Shh-induced tumors (for example, lung cancer, chondrosarcomas, etc.) and other tumors (for example, breast cancer, genitourinary cancer (for for example, kidney, bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinal cancer (for example, stomach, intestine, etc.), etc.).

In yet another embodiment of the present invention, compositions comprising hedgehog antagonists can be used in the in vitro generation of skeletal tissue, such as from skeletal stem cells, as well as in the in vivo treatment of skeletal tissue deficiencies. The present invention particularly contemplates the use of hedgehog antagonists to regulate the rate of chondrogenesis and / or osteogenesis. By "skeletal tissue deficiency" is meant a deficiency in the bone or other connective tissue of the skeleton at any site where it is desired to restore the bone or connective tissue, regardless of how the deficiency originates, for example, either as a result of surgical intervention, removal of a tumor, ulcer, implant, fracture or other traumatic or degenerative states.

For example, the method of the present invention can be used as part of a regime to restore function of cartilage for connective tissue. Such methods are useful, for example, in the repair of defects or injuries in the tissue of  cartilage that is the result of degenerative wear, such as which results in arthritis, as well as in other disorders mechanics that can be caused by tissue trauma, such as a displacement of the broken meniscus tissue, meniscectomy, a dislocation of a joint by a broken ligament, malignancy in the joints, bone fracture or hereditary disease. He present reparative method is also useful for remodeling the matrix of cartilage, such as in plastic or reconstructive surgery, as well as periodontal surgery The present method can also be applied to improve a previous reparative procedure, by example, after surgical repair of a meniscus, ligament, or cartilage. In addition, you can avoid the onset or exacerbation of a degenerative disease if applied soon enough after trauma

In one embodiment of the present invention, the object method comprises treating the affected connective tissue with a therapeutically sufficient amount of a hedgehog antagonist, particularly a selective antagonist for the Indian hedgehog transduction signal, to regulate a cartilage repair response in connective tissue controlling the rate of differentiation and / or proliferation of chondrocytes embedded in the tissue. Such connective tissues such as articular cartilage, interarticular cartilage (meniscus), costal cartilage (which connects the true ribs and the sternum), ligaments and tendons are particularly susceptible to treatment in reconstructive and / or regenerative therapies using the object method. As used herein, regenerative therapies include the treatment of degenerative states that have progressed to the point where tissue alteration is obviously manifest, as well as preventive tissue treatments in which degeneration is in their initial phases or is imminent.

In an illustrative embodiment, the object method can be used as part of a therapeutic intervention in the cartilage treatment of a diarthodic joint, such as one knee, one ankle, one elbow, one hip, one wrist, one knuckle of a finger or toe, or a joint Tempomandibular The treatment can be directed to the meniscus of the joint, articular cartilage of the joint, or both. To further illustrate, the object method can be used to treat a degenerative disorder of a knee, such as the one it could be the result of a traumatic injury (for example, a sports injury or excessive wear) or osteoarthritis. The Objective antagonists can be administered as an injection into the joint with, for example, an arthroscopic needle. In some cases, the injected agent may be in the form of a hydrogel or another slow release vehicle described above, in order to allow more prolonged and regular contact of the agent with the treated tissue

The present invention further contemplates the use of object method in the field of cartilage transplantation and treatments with a prosthetic device. However, they arise problems, for example, because the characteristics of the cartilage and fibrocartilage vary between different tissues, such as between articular, meniscus cartilage, ligaments and tendons, between the two ends of the same ligament or tendon, and between the superficial and deep parts of the tissue. The Zonal arrangement of these tissues may reflect a gradual change in mechanical properties and failure occurs when tissue implanted, which has not differentiated under those conditions, has no ability to respond appropriately. For example, when used meniscus cartilage to repair anterior cruciate ligaments, The tissue undergoes a metaplasia to pure fibrous tissue. Regulating the chondrogenesis rate, the object method can be used to address particularly this problem, helping to adaptively control cells implanted in the new environment and resembling effectively to hypertrophic chondrocytes of a phase of early tissue development

In a similar way, the object method can be applied to enhance both the generation of prosthetic cartilage devices and their implants. The need for improved treatment has motivated research aimed at creating new cartilage based on collagen-glucosaminoglycan molds (Stone et al . (1990) Clin Orthop Relat Red 252: 129), isolated chondrocytes (Grande et al . (1989 ) J Orthop res 7: 208; and Takigawa et to the (1987) Bone Miner 2:. 449) and chondrocytes attached to natural or synthetic polymers (Walitani et to the (1989) J Bone Jt Surg 71B:. 74; Vacanti et to the ( 1991) Plast Reconstr Surg 88: 753; von Schroeder et al . (1991) J Biomed Mater Res 25: 329; Freed et al . (1993) J Biomed Mater Res 27:11; and US Pat. to Vacanti et al . number 5,041,138). For example, chondrocytes can be grown in culture in biodegradable, biocompatible, highly porous structures, formed from polymers such as poly (glycolic acid), poly (lactic acid), agarose gel or other polymers that degrade over time in function of the hydrolysis of the polymer skeleton in harmless monomers. The matrices are designed to allow the proper exchange of nutrients and gases with the cells until the graft is produced. Cells can be cultured in vitro until adequate cell volume and density have been developed for cells to implant. An advantage of the matrices is that they can be cast or molded into a desired shape individually, so that the final product closely resembles the patient's own ear or nose (by way of example), or flexible matrices that allow manipulation at the time of implantation, as in a joint.

In one embodiment of the object method, the implants are contacted with a hedgehog antagonist during certain phases of the culture process, in order to control the rate of differentiation of the chondrocytes and the formation of hypertrophic chondrocytes in the culture.

In another embodiment, the implanted device is treated with a hedgehog antagonist in order to actively reshape the implanted matrix and make it more suitable for the desired function. As explained above with respect to tissue transplants, artificial transplants experience the same deficiency of not deriving in an environment that is comparable to the actual mechanical environment in which the matrix is implanted. The ability to regulate chondrocytes in the matrix by the object method may allow the implant to acquire characteristics similar to those of the tissue to which it is desired to replace.

In yet another embodiment, the object method is used to enhance the union of prosthetic devices. For To illustrate, the object method can be used in the implementation of a periodontal prosthesis, in which the treatment of connective tissue surrounding stimulates periodontal ligament formation around of the prosthesis.

In still further embodiments, the object method can be employed as part of a regimen for the generation of bone (osteogenesis) at a site in the animal where such skeletal tissue is deficient. Indian hedgehog is particularly associated with hypertrophic chondrocytes that are eventually replaced by osteoblasts. For example, the administration of a hedgehog antagonist of the present invention can be employed as part of a method to regulate the rate of bone loss in a subject. For example, preparations comprising hedgehog antagonists may be employed, for example, to control endochondral ossification in the formation of a "model" for ossification.

In yet another embodiment of the present invention, a hedgehog antagonist can be used to regulate spermatogenesis. It has been shown that hedgehog proteins, particularly Dhh, involved in differentiation and / or proliferation and maintenance of testicular germ cells. Dhh expression begins in precursors of the Sertoli cell shortly after the activation of Sry (testicular determinant gene) and persists in the testicles in the adult. Males are viable but sterile, due to a complete absence of mature sperm. Examination of the developing testicles in different genetic backgrounds suggests that Dhh regulates the early and late stages of spermatogenesis. Bitgood et al . (1996) Curr Biol 6: 298. In a preferred embodiment, the hedgehog antagonist can be used as a contraceptive. Similarly, hedgehog antagonists of the object method are potentially useful for modulating normal ovarian function.

The object method also has wide applicability for the treatment or prophylaxis of disorders affecting epithelial tissue, as well as in cosmetic uses. In general, the method can be characterized as including a step of administering to an animal an amount of a hedgehog antagonist effective to modify the growth state of a treated epithelial tissue. The mode of administration and dosage regimens will vary depending on the epithelial tissue (s) to be treated. For example, topical formulations will be preferred when the treated tissue is epidermal tissue, such as dermal or mucous tissues.

A method that "enhances the healing of a wound "results in the wound healing faster as treatment result, from what a similar wound takes in Heal in the absence of treatment. "Stimulation of healing of wounds "may also mean that the method regulates the proliferation and / or growth of, among others, keratinocytes, or that the wound heals with less scarring, less contraction of the wound, less collagen deposition and more surface area. In certain cases, "stimulation of wound healing" It can also mean that certain wound healing methods their success rates have improved (for example, the rates taken from skin grafts), when used together with the method of present invention

Despite significant progress in reconstructive surgical techniques, healing can be a major obstacle in the recovery of normal function and appearance of cured skin. This is particularly true when pathological scarring, such as keloids or hypertrophic scars of the hands or face produce functional disability or physical deformity. In the most serious circumstances, such scarring can push psychosocial difficulties and a life of economic hardship. Wound repair includes the phases of hemostasis, inflammation, proliferation and remodeling. The proliferative phase includes the multiplication of fibroblasts and endothelial and epithelial cells. By using the object method, the rate of proliferation of epithelial cells in and near the wound can be controlled in order to accelerate wound closure and / or minimize tissue formation.
scar.

The present treatment can also be effective as part of a therapeutic regimen for treating oral and paraoral ulcers, for example, resulting from radiation and / or chemotherapy. Such ulcers commonly develop within days after chemotherapy or radiation treatment. These ulcers usually begin as small, painful, irregularly shaped lesions, normally covered by a thin gray necrotic membrane and surrounded by inflammatory tissue. In many cases, lack of treatment results in the proliferation of tissue around the periphery of the lesion in an inflammatory manner. For example, the epithelium surrounding the ulcer normally demonstrates proliferative activity, which results in loss of continuity of the superficial epithelium. These lesions, due to their size and loss of epithelial integrity, predispose the organism to a potential secondary infection. The usual ingestion of food and water becomes a very painful event and, if ulcers proliferate throughout the digestive tract, diarrhea usually manifests with all its complication factors. According to the present invention, a treatment for such ulcers that includes the application of a hedgehog antagonist can reduce the abnormal proliferation and differentiation of the affected epithelium, helping to reduce the severity of subsequent inflammatory events.

The method and compositions object also can be used to treat wounds that result from diseases dermatological, such as lesions that result from disorders autoimmune, such as psoriasis. Atopic dermatitis is refers to skin trauma resulting from associated allergies with an immune response produced by allergens such as pollen, food, dandruff, insect poisons and toxins from plants.

In other embodiments, hedgehog antagonist antiproliferative preparations can be used to inhibit epithelial cell proliferation in the lens to avoid postoperative complications of extracapsular cataract extraction. Cataracts is a treatment-resistant eye disease and various studies have been conducted on a treatment of cataracts. However, at present, the treatment of cataracts is achieved through surgical interventions. Cataract surgery has been applied for a long time and various surgical methods have been examined. Extracapsular lens extraction has become the method of choice to remove cataracts. The main medical advantages of this technique with respect to intracapsular extraction are the lower incidence of aphakic cystoid macular edema and retinal detachment. Extracapsular extraction is also required for the implantation of intraocular lenses of the posterior chamber type that are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the high incidence of opacification of the posterior lens capsule, often referred to as after-cataract ("after-cataract"), which can occur in up to 50% of cases in within three years after surgery. The trans-cataract is produced by the proliferation of epithelial cells of the anterior and equatorial capsule of the lens that remain after extracapsular extraction of the lens. These cells proliferate to produce Sommerling rings, and together with the fibroblasts that are also deposited and produced in the posterior capsule, produce opacification of the posterior capsule, which interferes with vision. The prevention of trans-cataract would be preferable to treatment. To inhibit the formation of secondary cataracts, the object method provides a means to inhibit the proliferation of the remaining epithelial cells of the lens. For example, such cells can be induced to remain quiescent by instilling a solution containing a hedgehog antagonist preparation in the anterior chamber of the eye after lens extraction. In addition, the solution can be osmotically balanced to provide the minimum effective dose when instilled in the anterior chamber of the eye, thus inhibiting subcapsular epithelial growth with some specificity.

The object method can also be used in the treatment of corneopathies characterized by proliferation of keratinocytic epithelial cells, such as in the ocular epithelial disorders, such as epithelial growth inward or surface squamous cell carcinomas ocular.

Levine et al . (1997) J Neurosci 17: 6277 show that hedgehog proteins can regulate the mitogenesis and differentiation of photoreceptors in the vertebrate retina and that Ihh is a candidate factor of the pigmented epithelium to enhance the proliferation of retinal progenitors and the differentiation of The photoreceptors Likewise, Jensen et al . (1997) Development 124: 363 demonstrated that treatment of cultures of perinatal mouse retinal cells with the amino-terminal fragment of the Sonic hedgehog protein resulted in an increase in the proportion of cells incorporating bromodeoxyuridine, in the number of cells total, and in cane photoreceptors, amacrine cells and Muller's neurogliocytes, suggesting that Sonic hedgehog enhances the proliferation of retinal precursor cells. Therefore, the object method can be used in the treatment of proliferative diseases of retinal cells and to regulate the differentiation of photoreceptors.

Still another aspect of the present invention is refers to the use of the object method to control the growth of hair. The hair is basically made up of keratin, a resistant and insoluble protein; its main resistance lies in its cystine disulfide bridge. Each individual hair comprises a cylindrical axis and a root, and is contained in a follicle, a lageniform depression in the skin. The bottom of the follicle it contains a finger-shaped projection called papilla, which it consists of connective tissue from which the hair grows, and to through which blood vessels supply nutrients to the cells. The axis is the part that extends out of the skin surface, while the root has been described as the Inserted part of the hair. The root base extends to the hair bulb, resting on the papilla. Starting cells from which the hair is produced they grow in the follicle bulb; be they extrude in the form of fibers as cells proliferate in the follicle The "growth" of the hair refers to the formation and elongation of hair fiber by cells that they divide themselves.

As is well known in the art, the cycle Common hair is divided into three phases: anagen, catagen and telogen During the active (anagen) phase, the stem cells Epidermal dermal papilla divide rapidly. The cells daughter move up and differentiate to form the layers concentric of the hair itself. The transition phase, catagen, is characterized by the cessation of mitosis of stem cells in the follicle. The resting phase is known as telogen, in which the hair stays inside the scalp for several weeks before a new emerging hair that develops below, move the axis of the telogen phase of your follicle. From This model has made it clear that the larger the set of dividing stem cells that differ in hair cells, more hair growth occurs. Consequently, they can lead to Out methods to increase or reduce hair growth by potentiation or inhibition, respectively, of the proliferation of these stem cells.

In certain embodiments, the object method can be used as a way to reduce the growth of human hair, as opposed to conventional removal by cutting, shaving or hair removal. For example, the present method can be used in the treatment of trichosis, characterized by abnormally rapid or dense hair growth, for example, hypertrichosis. In an example embodiment, hedgehog antagonists can be used to treat hirsutism, a disorder characterized by abnormal hairiness. The object method can also provide a process to prolong the duration of hair removal.

In addition, since a hedgehog antagonist will often be cytostatic for epithelial cells, rather than cytotoxic, such agents can be used to protect hair follicle cells from cytotoxic agents that require progression in the S phase of the cell cycle for their effectiveness, for example, radiation induced death. Treatment by the object method can provide protection by causing the hair follicle cells to become quiescent, for example, by inhibiting the cells from entering the S phase and, therefore, preventing the follicle cells from suffering mitotic catastrophe or programmed cell death. . For example, hedgehog antagonists can be used for patients who undergo chemotherapies or radiotherapies, which usually result in hair loss. By inhibiting the progression of the cell cycle during such therapies, the object treatment can protect the hair follicle cells from death which, otherwise, could result from the activation of cell death programs. Upon completion of the treatment, the present method can also be eliminated with the concomitant relief of inhibition of follicle cell proliferation.

The object method can also be used in the treatment of folliculitis, such as decalvating folliculitis, reticulated uleritematous folliculitis and keloid folliculitis. For example, a cosmetic preparation of a hedgehog antagonist can be applied topically in the treatment of pseudofolliculitis, a chronic disorder that occurs most frequently in the submandibular region of the neck and is associated with shaving, whose characteristic lesions are pustules and Erythematous papules containing inserted hairs.

In another aspect of the invention, the object method can be used to induce differentiation and / or to inhibit the proliferation of epithelially derived tissue. Such forms of these molecules can provide a basis for the therapy of differentiation for the treatment of hyperplastic states and / or Neoplastic involving epithelial tissue. For example, such Preparations can be used for the treatment of skin diseases in which there is a proliferation or growth abnormal skin cells.

For example, pharmaceutical preparations of the invention are directed to the treatment of epidermal states hyperplastic, such as keratosis, as well as for treatment of neoplastic epidermal states, such as those characterized by a high proliferation rate of various skin cancers, such as squamous cell carcinoma. The method object can also be used in the treatment of diseases autoimmune diseases that affect the skin, in particular, of dermatological diseases that include keratinization and / or morbid proliferation of the epidermis, such as produced by psoriasis or atopic dermatosis.

Many common skin diseases, such such as psoriasis, squamous cell carcinoma, the keratoacanthoma and actinic keratosis, are characterized by localized anomalous proliferation and growth. For example, in the  psoriasis, which is characterized by scaly, red and elevated skin, keratinocytes are known to proliferate much faster than normal and they differ less completely.

In one embodiment, the preparations of the present invention are suitable for the treatment of dermatological conditions associated with keratinization disorders that produce abnormal skin cell proliferation, disorders that can be characterized by inflammatory or non-inflammatory components. As an illustration, therapeutic preparations of a hedgehog antagonist can be used, for example, that enhances quiescence or differentiation, to treat various forms of psoriasis, whether cutaneous, mucosal or nail. Psoriasis, as described above, is typically characterized by epidermal keratinocytes that have marked proliferative activation and differentiation along a "regenerative" pathway. Treatment with an antiproliferative embodiment of the object method can be used to reverse pathological epidermal activation and can provide a basis for sustained disease remission.

A variety of keratotic lesions are also candidates for treatment with the object method. Actinic keratoses, for example, are superficial inflammatory premalignant tumors that arise from irradiated and sun-exposed skin. The lesions are from erythematous to brown with variable scaling. Current treatments include cryosurgery and excisional surgery. However, these treatments are painful and often produce aesthetically unacceptable scars. Accordingly, the treatment of keratosis, such as actinic keratosis, may include the application, preferably topical, of a hedgehog antagonist composition in amounts sufficient to inhibit hyperproliferation of the epidermal / epidermoid cells of the lesion.

Acne represents yet another dermatological condition that can be treated by the object method. Acne vulgaris , for example, is a multifactorial disease that occurs most commonly in adolescents and young adults, and is characterized by the appearance of inflammatory and non-inflammatory lesions on the face and upper trunk. The basic defect that gives rise to acne vulgaris is hypercornification of the duct of an overactive sebaceous gland. Hypercornification blocks the normal mobility of the microorganisms of the skin and follicles, and in doing so, stimulates the release of lipases by the bacteria Propinobacterium acnes and Staphylococcus epidermidis and the yeast Pitosporum ovale . Treatment with an antiproliferative hedgehog antagonist, particularly in topical preparations, may be useful to avoid the transition characteristics of the ducts, for example, hypercornification, which lead to the formation of the lesion. The subject treatment may also include, for example, antibiotics, retinoids and antiandrogens.

The present invention also provides a method to treat various forms of dermatitis. Dermatitis is a descriptive term that refers to injuries sparingly delimited that are pruritic, erythematous, flaky, vesiculous, suppurating, cracked or crusted. These injuries for any of a wide variety of causes. The most types Common dermatitis are atopic, contact, and dermatitis diaper. For example, seborrheic dermatitis is a dermatitis chronic, usually pruritic, with erythema, scale formation dry, wet or fatty, and patches with yellow crust in various areas, especially in the scalp, with exfoliation of a excessive amount of dry scales. The object method can also be used in the treatment of stasis dermatitis, a often chronic dermatitis, usually eczematous. Dermatitis Actinic is dermatitis due to radiation exposure actinic, such as from the sun, ultraviolet waves or X or gamma radiation. According to the present invention, the method object can be used in the treatment and / or prevention of certain symptoms of dermatitis caused by proliferation not Desired epithelial cells. Such therapies for these various forms of dermatitis can also include corticosteroids, antipuritics and antibiotics, topical and systemic

The ailments that can be treated by object method are specific disorders for non-human beings, such as scabies.

In yet another embodiment, the subject method can also be used in the treatment of human cancers, such as epithelial tissue tumors, such as the skin. For example, hedgehog antagonists can be used, in the object method, as part of a treatment for carcinomas, adenocarcinomas, human sarcomas, and the like.

In another aspect, the present invention provides pharmaceutical preparations comprising hedgehog antagonists. Hedgehog antagonists for use in the subject method can be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures of the same. The optimum concentration of the active ingredient (s) in the chosen medium can be determined empirically, according to procedures well known to pharmaceutical chemists. As used herein, "biologically acceptable medium" includes any of all solvents, dispersion media and the like, which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such means for pharmaceutically active principles is known in the art. Except as long as any conventional media or agent is incompatible with the activity of hedgehog antagonists, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their included formulation of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "tank formulations."

The pharmaceutical formulations of the present invention also include veterinary compositions, for example, pharmaceutical preparations of hedgehog antagonists suitable for veterinary uses, for example, for the treatment of livestock or domestic animals, for example, dogs.

Introduction methods can also be provided by rechargeable or biodegradable devices. Several polymeric slow-release devices have been developed and tested in vivo in recent years for the controlled administration of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including biodegradable and non-degradable polymers, can be used to form an implant for sustained release of a hedgehog antagonist at a particular target site.

The preparations of the present invention may administered orally, parenterally, topically or rectally. Naturally, they are administered by appropriate forms for each route of administration For example, they are administered in the form of tablets or capsules, by injection, inhalation, lotion eyepiece, ointment, suppository, controlled release patch, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal through suppositories. The Oral and topical administrations are preferred.

The expressions "parenteral administration" and "administered parenterally," as used in the This document means modes of administration other than enteric or topical administration, usually by injection, and include, without limitation, intravenous injection or infusion, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal.

The expressions "systemic administration", "systemically administered", "administration peripheral "and" administered peripherally, "as  used herein, means the administration of a compound, drug or other material, other than directly in the central nervous system, so that it enters the system of the patient and, therefore, undergo metabolism and other processes similar, for example, subcutaneous administration.

These compounds can be administered to beings humans and other animals for treatment by any proper route of administration, including orally, nasally, for example, a sprayer, rectally, intravaginal, parenteral, intracisternal, and topically, by  powders, ointments or drops, including orally and sublingually.

Regardless of the route of administration selected, the compounds of the present invention, which can be used in a suitable hydrated form, and / or the compositions Pharmaceuticals of the present invention are formulated in forms Pharmaceutically acceptable pharmaceuticals, as described further forward, or by other conventional methods known to experts in the art.

The actual dose levels of the principles active in the pharmaceutical compositions of this invention may varied so that an amount of active ingredient is obtained that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without Be toxic to the patient.

The dose level selected depends on a variety of factors, including compound activity particular of the present invention employed, or of the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound that is being used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular hedgehog antagonist employed, age, sex, weight, status, general health and previous medical history of the patient who it is being treated, and similar factors well known in the medical techniques

A doctor or veterinarian who is an expert usual in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, the doctor or veterinarian could start with doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than required, in order to achieve the effect therapeutic desired and gradually increase the dose until it Achieve the desired effect.

In general, an adequate daily dose of a compound of the invention will be that amount of the compound that be the lowest effective dose to produce a therapeutic effect. Such an effective dose will generally depend on the factors described. previously. Generally, intravenous doses, intracerebroventricular and subcutaneous compounds of this invention for a patient will range from about 0.0001 up to about 100 mg per kilogram of body weight per day.

If desired, the effective daily dose of the compound asset can be managed as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in pharmaceutical forms unitary

The term "treatment" is intended to encompass also prophylaxis, therapy and cure.

The patient receiving this treatment is any animal that needs it, including primates, in particular humans, and other mammals such as horses, cattle, pigs and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or in admixture with pharmaceutically acceptable vehicles and / or sterile and can also be given together with other agents antimicrobials such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Therefore, the joint treatment includes sequential, simultaneous and separate administration of active compound in a way that the therapeutic effects of first administered don't disappear completely when you Manage the following.

V. Pharmaceutical compositions

Although it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The hedgehog antagonists according to the invention can be formulated for administration in any convenient form for use in human and veterinary medicine. In certain embodiments, the compound included in the pharmaceutical preparation may be active on its own, or it may be a prodrug, for example, capable of becoming an active compound in a physiological environment.

Therefore, another aspect of the present invention provides pharmaceutically acceptable compositions that they comprise a therapeutically effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. As described in detail below, the compositions Pharmaceuticals of the present invention can be formulated especially for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, impregnations (drains) (solutions or suspensions aqueous or non-aqueous), tablets, bowling, powders, granules, pastes for application on the language; (2) parenteral administration, by example, by subcutaneous, intramuscular or intravenous injection, such as a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginal route or intrarectal, for example, as a vaginal ovum, cream or foam. However, in certain embodiments, the subject compounds may dissolve or suspend simply in sterile water. In certain embodiments, the pharmaceutical preparation is non-pyrogenic, it is That is, it does not raise a patient's body temperature.

The term "therapeutically effective amount", as used herein, means such an amount of a compound, material or composition that comprises a compound of the present invention that is effective in producing a certain desired therapeutic effect by overcoming a gain phenotype of hedgehog function in at least one subpopulation of cells in an animal and thus blocking the biological consequences of that route in the treated cells, in a reasonable benefit / risk ratio applicable to any medical treatment.

The expression "pharmaceutically acceptable" is used in this document to refer to those compounds, materials, compositions and / or pharmaceutical forms that are, within the scope of solid medical criteria, suitable for its use in contact with the tissues of humans and animals without toxicity, irritation, allergic response or other problems or excessive complications, according to a benefit / risk ratio reasonable.

The expression "pharmaceutical vehicle acceptable ", as used herein, means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulation material, involved in carrying or transport the antagonists object of an organ, or part of the organism, to another organ, or part of the organism. Each vehicle must be "acceptable" in the sense of being compatible with others formulation components and not harmful to the patient. Some examples of materials that can serve as vehicles Pharmaceutically acceptable include: (1) sugars, such as lactose, glucose and sucrose; (2), starches, such as starch corn and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (5) jelly; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, seed oil cotton, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (19) Ringer solution; (19) ethyl alcohol; (20) buffer solutions phosphate; and (21) other compatible non-toxic substances used in pharmaceutical formulations.

As explained above, certain embodiments of the present hedgehog antagonists may contain a basic functional group, such as amino or alkylamino and, therefore, may form pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this sense refers to relatively non-toxic organic or inorganic acid addition salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by reacting separately a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus. formed. Representative salts include salts hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate and lauryl sulphonate and the like. (See, for example, Berge et al . (1977) "Pharmaceutical Salts", J. Pharm. Sci . 66: 1-19).

The pharmaceutically acceptable salts of subject compounds include conventional non-toxic salts or salts quaternary ammonium compounds, for example, from non-toxic organic or inorganic acids. For example, such salts Conventional non-toxic include those derived from acids inorganic such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared to from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxyloleic, phenylacetic, glutamic, benzoic, salicylic, sulphanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isotionic and Similar.

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, therefore, may form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts", in these cases, refers to relatively non-toxic organic and inorganic base addition salts of the compounds of the present invention. These salts can also be prepared in situ during the final isolation and purification of the compounds of the invention, or by reacting separately a purified compound in its free acid form with a suitable base such as hydroxide, carbonate or bicarbonate, of a cation Pharmaceutically acceptable metal, with ammonia or with a pharmaceutically acceptable primary, secondary or tertiary organic amine. Representative alkaline or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al ., Cited above).

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and stearate magnesium, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents and flavorings, preservatives and antioxidants, may also be present in the compositions.

Examples of antioxidants pharmaceutically Acceptable include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2). oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and Similar; and (3) metal chelating agents, such as acid citric, ethylenediaminetetraacetic acid (EDTA), sorbitol, acid tartaric, phosphoric acid, and the like.

The formulations of the present invention include those suitable for oral, nasal, topical (including oral and sublingual), rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form or may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce an individual pharmaceutical form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce an individual pharmaceutical form will generally be that amount of the compound that produces a therapeutic effect. In general, from one hundred percent, this amount will range from about 1 percent to about ninety-nine percent active ingredient, preferably from about 5 percent to about 70 percent, more preferably from about 10 percent. one hundred to about 30 per
hundred.

The methods to prepare these formulations or Compositions include the step of associating a compound of the present invention with the vehicle and, optionally one or more auxiliary components In general, the formulations will be prepared uniformly and intimately associating a compound of the present invention with liquid vehicles, or finely solid vehicles divided, or both, and then, if necessary, shape the product.

The formulations of the invention suitable for oral administration may be in the form of capsules, sachets, pills, tablets, pills (using a sweetened base, normally sucrose and gum arabic or tragacanth), powders, granules or as a solution or a suspension in an aqueous liquid or not aqueous, or as a liquid emulsion of oil in water or water in oil, or as an elixir or syrup, or as pills (using a inert base, such as gelatin and glycerin, or sucrose and gum arabic) and / or as mouthwashes and the like, containing each a predetermined amount of a compound of the present invention as active ingredient. A compound of the present invention can also be administered as a bolus, electuary or pasta.

In the solid pharmaceutical forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active substance is mixture with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato starch or tapioca, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as ammonium compounds quaternary; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, polyethylene glycols solids, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents In the case of capsules, tablets and pills, pharmaceutical compositions may also comprise agents buffers Solid compositions of a similar type also can be used as filters in loaded gelatin capsules hard and soft that use excipients such as lactose or milk sugars, as well as high-weight polyethylene glycols molecular and the like.

A tablet can be obtained by compression or molding, optionally with one or more auxiliary components. The Compressed tablets can be prepared using a binding agent (for example, gelatin or hypromellose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or sodium carboxymethyl cellulose crosslinked), surfactant or dispersant. Molded tablets they can be obtained by molding in a suitable machine a mixture of the powder compound moistened with an inert liquid diluent.

Tablets and other pharmaceutical forms solids of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, can be marked optionally or be prepared with coatings and envelopes, such as enteric coatings and other coatings well known in the Pharmaceutical formulation technique They can also be formulated from so that they provide slow or sustained release from the beginning active using in it, for example, hypromellose in various proportions to provide the desired release profile, other polymeric matrices, liposomes and / or microspheres. They can sterilized, for example, by filtration through a filter that retains bacteria, or by incorporating sterilizing agents in the form of solid compositions sterile that can dissolve in sterile water, or some other medium Sterile injection immediately before use. These compositions may also optionally contain agents opacifying and can be of a composition in which they release the active principle (s) only, or preferably, in a certain part of the gastrointestinal tract, optionally in a sustained manner. Examples of compositions of Inclusions that can be used include polymeric substances and waxes The active substance may also be in shape. microencapsulated, if appropriate, with one or more of the excipients described above.

Liquid pharmaceutical forms for Oral administration of the compounds of the invention include emulsions, microemulsions, solutions, suspensions, syrups and elixirs, pharmaceutically acceptable. In addition to the active substance, Liquid dosage forms may contain diluents. inert commonly used in the art, such as, for example, water and other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed oils, peanut, corn, olive, castor and sesame), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof.

In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavorings, colorants, flavorings and agents preservatives

The suspensions, in addition to the compounds assets, may contain suspending agents such as, for for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, cellulose microcrystalline, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

It is known that sterols, such as cholesterol will form complexes with cyclodextrins. Therefore in preferred embodiments, when the inhibitor is an alkaloid Steroid, can be formulated with cyclodextrins, such as α, β and γ-cyclodextrin, dimethyl-? -cyclodextrin and 2-hydroxypropyl-? -Cyclodextrin.

The formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients and vehicles comprising, for example, butter cocoa, polyethylene glycol, a suppository wax or a salicylate, and that is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active hedgehog antagonist.

The formulations of the present invention that are suitable for vaginal administration also include vaginal egg formulations, buffers, creams, gels, pastes, foams or sprays containing vehicles such as It is known in the art that they are appropriate.

Appropriate pharmaceutical forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalations. He active compound can be mixed under sterile conditions with a pharmaceutically acceptable vehicle and with any preservative, buffer or propellant that may be required.

Ointments, pastes, creams and gels can contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Dusts and sprays may contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, silicates calcium and polyamide powder, or mixtures of these substances. The sprays may additionally contain propellants common, such as chlorofluorohydrocarbons and hydrocarbons Unsubstituted volatiles, such as butane and propane.

Transdermal patches have the added advantage of providing controlled administration of a compound of the present invention to the body. Such pharmaceutical forms can be obtained by dissolving or dispersing hedgehog antagonists in the appropriate medium. Absorption enhancers can also be used to increase the flow of hedgehog antagonists through the skin. The speed of such flow can be controlled by providing a velocity control membrane or by dispersing the compound in a gel or polymer matrix.

Ophthalmic formulations, ointments, powders, ocular solutions and the like, it is also contemplated that they are within the scope of this invention.

The pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more solutions, dispersions, suspensions or emulsions, aqueous or not aqueous, isotonic, sterile, pharmaceutically acceptable, or sterile powders that can be reconstituted in solutions or sterile injectable dispersions just before use, which may contain antioxidants, buffers, bacteriostatics, solutes that make the formulation isotonic with the blood of desired receptor or suspending agents or thickeners.

Examples of aqueous and non-aqueous vehicles suitable that can be used in the pharmaceutical compositions of The invention includes water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and mixtures of same, vegetable oils, such as olive oil, and esters injectable organics, such as ethyl oleate. The fluidity appropriate can be maintained, for example, by using coating materials such as lecithin, by means of the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The prevention of the action of microorganisms can be guaranteed by the inclusion of various antifungal and antibacterial agents, for example, paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and
jelly.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of Subcutaneous or intramuscular injection drug. This can be carried out by using a liquid suspension of crystalline or amorphous material that have poor water solubility. The absorption rate of the drug then depends on its speed of dissolution which, in turn, may depend on the size of the crystal and of the crystalline form. Alternatively, delayed absorption in a parenterally administered pharmaceutical form it is carried carried out by dissolving or suspending the drug in a vehicle oily

Injectable extended release forms are obtained by forming microencapsulated matrices of the compounds object in biodegradable polymers, such as polylactide-polyglycolide. Depending on the reason of the drug with respect to the polymer, and of the nature of the particular polymer employed, the rate of drug release Examples of other biodegradable polymers they include poly (orthoesters) and poly (anhydrides). The Injectable extended-release formulations are also they prepare by trapping the drug in liposomes or microemulsions that are compatible with body tissue.

When the compounds of the present invention are administer as pharmaceutical products to humans and animals, they can be administered per se or as a pharmaceutical composition that it contains, for example, 0.1 to 99.5% (more preferably, of 0.5 to 90%) of the active substance in combination with a vehicle pharmaceutically acceptable.

The addition of the active compound of the invention to the feeding of the animal is preferably carried out preparing an appropriate food premix containing the active compound in an effective amount and incorporating the premix to the full ration.

Alternatively, it can be mixed with food an intermediate concentrate or supplementary food intake that They contain the active substance. The way in which such premixes food and whole rations can be prepared and administered are described in bibliography books (such as "Applied Animal Nutrition ", W.H. Freedman and CO., San Francisco, USA, 1969 or "Livestock Feeds and Feeding" O and B Books, Corvallis, Pray., USA, 1977).

SAW. Synthetic schemes and antagonist identification assets

The object steroidal alkaloids, and congeners of these, can easily be prepared using technologies cross coupling of Suzuki, Stille and the like. These coupling reactions are carried out under conditions relatively soft and tolerate a wide variety of functionality "spectator."

to. Combinatorial libraries

The compounds of the present invention, particularly libraries or variants having several representative classes of substituents, can be treated by combinatorial chemistry and other parallel synthesis schemes (see, for example, PCT WO 94/08051). The result is that large libraries of related compounds can be rapidly selected, for example, a motley library of the compounds depicted above, in high performance assays in order to identify potential leading hedgehog antagonist compounds, as well as to refine specificity, toxicity and / or the cytotoxic-kinetic profile of a leader compound. For example, bioactivity tests of ptc, hedgehog or smoothened can be used, such as can be developed using cells with loss of ptc function, gain of hedgehog function or gain of smoothened function, to select a library of the subject compounds for which they have activity agonist against ptc or antagonistic activity against hedgehog or smoothened .

For illustration only, a library combinatorial for the purposes of the present invention is a mixture of chemically related compounds that can Select together for a desired property. The preparation of many related compounds in a single reaction reduces and greatly simplifies the number of selection processes that is necessary to carry out. The selection for physical properties Appropriate can be done by conventional methods.

You can create diversity in the library at a variety of different levels. For example, aryl groups substrate used in combination reactions can be different in regard to the central aryl moiety, for example, a diversity in regard to the structure of the ring, and / or may vary with respect to the other substituents.

A variety of technical resources are available in the art to generate combinatorial libraries of small organic molecules such as the object hedgehog agonists. See, for example, Blondelle et al . (1995) Trends Anal. Chem 14:83; U.S. patents granted to Affymax 5,359,115 and 5,362,899; U.S. Patent granted to Ellman 5,288,514; PCT publication granted to Still et al . WO 94/08051; U.S. patents granted to ArQule 5,736,412 and 5,712,171; Chen et al . (1994) JACS 116: 2661; Kerr et al . (1993) JACS 115: 252; PCT publications WO92 / 10092, WO93 / 09668 and WO91 / 07087; and the PCT publication granted to Lerner et al . WO93 / 20242). Accordingly, a variety of libraries of the order of about 100 to 1,000,000 or more divers of object hedgehog antagonists can be synthesized and selected for a particular activity or property.

In an exemplary embodiment, a library of candidate hedgehog antagonist diversomers can be synthesized using a scheme adapted to the techniques described in the PCT publication granted to Still et al . WO 94/08051, for example, which are attached to a polymeric bead by a hydrolysable or photolizable group, optionally located in one of the positions of the candidate antagonists or a substituent of a synthetic intermediate. According to the technique of Still et al ., The library is synthesized in a set of pearls, each pearl including a set of labels that identify the particular diversomers in that pearl. The pearl library can then be "plated" with cells with loss of ptc function, hedgehog function gain or smoothened function gain for which a hedgehog antagonist is sought. Diversomers can be released from the bead, for example, by hydrolysis.

The structures of the compounds useful in the The present invention easily lends itself to an effective synthesis. The nature of the structures, as generally described by formulas I and II, allows the assembly of such compounds using a certain combination of residues X, Y and Z, as explained above. The X and Z remains are of nature heteroatomic and therefore allow the formation of various carbon bonds. The remains And, when present, take the form of electrophilic moieties, such as carbonyl groups and sulfonyl, and reagents are readily available for binding of such moieties to heteroatomic groups similar to X and Z. The vast majority of such reactions, including those represented in figures 11, 12, 15 and 16 they are extremely soft and extremely reliable and therefore perfectly suited for combinatorial chemistry. The simplistic nature of such combinatorial approaches with respect to the generation of a library of test compounds is evident in the following example scheme (P = protective group), in which the various groups of a compound according to formula II are linked combinatorially (for example, using one of the methods described above), with combinatorial functionalization of central ring system (for example W) conferring diversity Additional to the library. You can get even more diversity, for example, using a variety of electrophilic groups when a subunit is added, that is, using a variety of PO-Ar-L-C (O) Cl, PO-Ar-L-NCO, PO-Ar-L-SO_ {Cl}, etc., when the R2 subunit is added.

17

Many variations of the above and related routes allow the synthesis of widely diverse libraries of compounds that can be tested as inhibitors of hedgehog function.

b. Selection Trials

A variety of assays are available to determine the ability of a compound to be an agonist of the ptc function or antagonist of the smoothened or hedgehog function, many of which may be available in high performance formats. In many drug selection programs that test libraries of natural compounds and extracts, high performance assays are desirable in order to maximize the number of compounds inspected in a given period of time. Therefore, libraries of natural and synthetic products can be sampled for other compounds that are hedgehog antagonists.

In addition to cell-free assays, test compounds can also be tested in cell-based assays. In one embodiment, the cell that has a phenotype of loss of ptc function, hedgehog function gain or smoothened function gain can be contacted with a test agent of interest, with the test score for, for example, inhibition of cell proliferation in the presence of the test agent.

Several gene products have been linked in the signal transduction mediated by patched , including patched , transcription factors of the cubitus interruptus (ci), fused (fu) family of serine / threonine kinase and costal-2 , smoothened and supressor of fused .

The induction of cells by hedgehog proteins sets in motion a cascade that involves the activation and inhibition of effectors downstream, whose final consequence is, in some cases, a detectable change in the transcription or translation of a gene. The potential transcriptional targets of hedgehog- mediated signaling are the patched gene (Hidalgo and Inhgam, 1990 Development 110, 291-201; Marigo et al ., 1996) and vertebrate homologs of the Drosophila cubitus interruptus gene , the GLI genes ( Hui et al . (1994) Dev Biol 162: 402-413). It has been shown that patched gene expression is induced in the cells of the yolk of the extremities and the neural plaque that is responsible for Shh . (Marigo et al . (1996) PNAS 93: 9346-51; Marigo et al . (1996) Development 122: 1225-1233). Gli genes encode for supposed transcription factors that have DNA binding zinc finger domains (Orenic et al . (1990) Genes & Dev 4: 1053-1067; Kinzler et al . (1990) Mol Cell Biol 10: 634 -642). It has been reported that the transcription of the Gli gene is regulated by an increase in response to hedgehog in the buds of the extremities, while the transcription of the Gli3 gene is regulated by a decrease in response to the induction of hedgehog (Marigo et al . (1996 ) Development 122: 1225-1233). By selecting transcriptional regulatory sequences from such target genes, for example, from the patched or Gli genes, which are responsible for the regulation by increase or decrease of these genes in response to hedgehog signaling, and which bind operationally such promoters to a reporter gene (reporter), a transcription based assay that is sensitive to the ability of a specific test compound to modify signaling pathways mediated by hedgehog can be obtained . Therefore, reporter gene expression provides a valuable screening tool for the development of compounds that act as hedgehog antagonists.

Assays based on the reporter gene of this invention measure the final phase of the cascade of events described above, for example, transcriptional modulation. Consequently, with the implementation of an embodiment of the assay, a reporter gene construct is inserted into the reactive cell in order to generate a detection signal that depends on the loss of ptc function, the hedgehog function gain, the smoothened function gain , or stimulation by SHH itself. The amount of transcription from the reporter gene can be measured using any method known to the person skilled in the art that is suitable. For example, mRNA expression from the reporter gene can be detected using RNAse or RNA-based PCR protection, or the protein product of the reporter gene can be identified by a characteristic staining or intrinsic biological activity. The amount of expression from the reporter gene is then compared to the amount of expression in the same cell in the absence of the test compound or can be compared to the amount of transcription in a substantially identical cell that lacks the target receptor protein. Any decrease in the amount of significant transcription from the statistical point of view or otherwise indicates that the test compound has functioned as an agonist in some way from the normal ptc signal (or has functioned as an antagonist of the hedgehog or smoothened gain signal function), for example, the test compound is a potential hedgehog antagonist.

Examples

The invention that is now being described generally, it will be more easily understood in reference to following examples that are included merely for the purpose of illustration of certain aspects and embodiments of this invention, and are not intended to limit the invention.

Lead compound discovery / high selection assay performance

The compounds to be tested are dissolved from DMSO to a concentration of 10 mM and stored at -20 ° C. To activate the Hedgehog pathway in the test cells, an octyl (lipid modified) form of the N-terminal fragment of the Sonic Hedgehog protein (OCT-SHH) is used. This N-terminal SHH fragment is produced by bacteria.

The compounds can be tested in the "Gli-Luc" test below, using the 10T cell line (s12), in which the cells contain a reporter construct that responds to Hedgehog using Luciferase as the reporter gene. In this way the activity of the Hedgehog signaling path can be measured by the Gli-Luc response.

10t1 / 2 cells (s12) are seeded in a 96 well microtiter plate (MTP) at 20,000 cells / well in complete medium [DMEM with 10% FBS]. Then, the plates were placed in the incubator for incubation overnight (O / N), at 37 ° C and 5% CO2. After 24 h, the medium is replaced with Luciferase assay medium (DMEM with the 0.5% FBS). The compounds are thawed and diluted in medium test at 3: 1000 (approximately 300 times) resulting in a starting concentration of about 30 µM.

Subsequently, 150 µl of each are added shows 30 µM in the first wells (in triplicate). The MTP samples are then diluted in dilutions of 3 times up to a total of seven wells, ultimately resulting in a regimen of seven dilutions in triplicate for each compound. TO OCT-SHH protein ligand is then diluted in the Luciferase assay medium and added to each well to a final concentration of 0.3 µg / ml. The plates are returned then to the incubator for further incubation O / N, at 37 ° C and the 5% of CO2. After approximately 24 h, the plates are removed from the incubator and the medium is aspirated / discarded. The wells are washed once with assay buffer [1 mM PBS + Mg 2+ and Ca 2+ 1 mM]. Then 50 µl of the assay buffer is added to each well. Luciferase test reagent is prepared as Describe the seller (Packard LucLite kit) and 50 \ mul are added to each well. The plates are incubated at room temperature (RT) for approximately 30 minutes after which the signal is read, from new to RT, on a TopCount device (Packard).

The compounds identified in this assay are represented in Figure 32. The discovery of these inhibitors of Shh-induced Gli transcription activity exemplifies the usefulness of the claims in this patent. The  activities of these compounds are presented in table 1 to continuation.

TABLE 1

18

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Additional information is presented in Figure 33. Mouse 456 is a heterozygous deficient in Ptc that received UV irradiation for 6 months. The mouse developed many lesions of small BCCs, which turned blue after staining by X-gal The mouse was sacrificed and the skin was removed with a 2 mm cutaneous punch. These cutaneous punches are They then cultivated for 6 days. Compared to the vehicle (DMSO), compound 11 may decrease the number of and size of BCC lesions (blue spots in the drawing). The effect was more obvious when 10 µM of 11 was added to the culture medium, instead of 5 µM of 11. In summary, this experiment suggests that  11 can inhibit murine BCC lesions in mouse number 456

In yet another experiment, lungs with lacc ptc-1 (dII) in stage E12.5 were collected and transgenic embryos were identified by lacZ detection using markers. The lung explants were grown submerged in mouse explant medium (based on DMEM, additives optimized for mouse lung culture) for 48 h, fixed in lacZ fixative, rinsed and stained for lacZ O / N at 37 ° C. The control tissue was not treated, while the test tissue was treated with 11. The results are depicted in Figure 34: (A) Untreated control. The strong expression of lacZ can be observed in the proximal and distal mesenchyme. (B) Treatment with 11 leads to a significant decrease in reporter gene expression, as evidenced especially by the weak signal surrounding the distal branching ends of the growing lung epithelium.

Preparation of compounds of the present invention to. Illustrative synthetic schemes

Example synthesis schemes for generating hedgehog antagonists useful in the methods and compositions of the present invention are shown in Figures 1-31.

The reaction conditions in the schemes Illustrated in Figures 1-31 are as follows:

one)

R 1 CH 2 CN, NaNH 2, Toluene

(Arzneim-Forsch, 1990, 40, 11, 1242)

2)

H 2 SO 4, H 2 O, Reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

3)

H 2 SO 4, EtOH, reflux

(Arzneim-Forsch, 1990, 40, 11, 1242)

4)

NaOH, EtOH, reflux

5)

(Boc) 2 O, ZM NaOH, THF

6)

LiHDMS, R1 X, THF

(application patent granted to Merck number WO 96/06609)

7)

Pd-C, H2, MeOH

8)

t-BuONO, CuBr, HBr, H2O

(J. Org. Chem. 1977, 42, 2426)

9)

ArB (OH) 2, Pd (PPh 3) 4, Dioxane

(J. Med. Chem. 1996, 39, 217-223)

10)

R 12 (H) C = CR 13 R 14, Pd (OAc) 2, Et 3 N, DMF

(Org. React. 1982, 27, 345)

eleven)

Tf 2 O, THF

(J. Am. Chem. Soc. 1987, 109, 5478-5486)

12)

ArSnBu_ {3}, Pd (PPh 3) 4, Dioxane

(J. Am. Chem. Soc. 1987, 109, 5478-5486)

13)

KMnO_ {4}, Py, H2O

(J. Med. Chem. 1996, 39, 217-223)

14)

NaOR1, THF

fifteen)

NaSR1, THF

16)

HNR 1 R 13, THF

17)

HONO, NaBF_ {4}

(Adv. Fluorine Chem. 1965, 4, 1-34)

18)

Pd (OAc) 2, NaH, DPPF, PhCH 3, R 1 OH

(J. Org. Chem. 1997, 62; 5413-5418)

19)

i. R 1 X, Et 3 N, CH 2 Cl 2, ii. R_ {13} X

twenty)

SOCl2, cat DMF

twenty-one)

CH 2 N 2, Et 2 O

22)

Ag 2 O, Na 2 CO 3, Na 2 S 2 O 3, H 2 O

(Tetrahedron Lett. 1979, 2667)

2. 3)

AgO 2 CPh, Et 3 N, MeOH

(Org. Syn., 1970, 50, 77; J. Am. Chem. Soc. 1987, 109, 5432)

24)

LiOH, THF-MeOH

25)

(EtO) 2 P (O) CH 2 CO 2 R, BuLi, THF

26)

MeO 2 CCH (Br) = P (Ph) 3, benzene

27)

Koh or KOtBu

28)

Base, X (CH 2) n, CO 2 R

29)

DPPA, Et3N, toluene

(Synthesis 1985, 220)

30)

HONO, H2O

31)

SO2, CuCl, HCl, H2O

(Synthesis 1969, 1-10, 6)

32)

Lawesson reagent, toluene

(Tetrahedron Asym. 1996, 7, 12, 3553)

33)

R2M, solvent

3. 4)

30% H 2 O 2, CH 3 CO 2 H glacial

(Helv. Chim. Minutes 1968, 349, 323)

35)

Triphosgene, CH 2 Cl 2

(Tetrahedron Lett., 1996, 37, 8589)

36)

i. (EtO) 2 P (O) CHLiSO 2 Oi-Pr, THF, ii. Nal

37)

Ph 3 PCH 3 l, NaCH 2 S (O) CH 3, DMSO

(Synthesis 1987, 498)

38)

Br 2, CHCl 3 or other solvent

(Synthesis 1987, 498)

39)

BuLi, Bu 3 SnCl

40)

ClSO_2 OTMS, CCl_ {}

(Chem. Ber. 1995, 128, 575-580)

41)

MeOH-HCl, Reflux

42)

THE H, Et 2 O or LiBH 4, EtOH or BH 3 -THF

(Tetrahedron Lett., 1996, 37, 8589)

43)

MsCl, Et3N, CH 2 Cl 2

(Tetrahedron Lett., 1996, 37, 8589)

44)

Na 2 SO 3, H 2 O

(Tetrahedron Lett., 1996, 37, 8589)

Four. Five)

R 2 R 4 NH, Et 3 N, CH 2 Cl 2

46)

R2M, solvent

47)

CH 3 NH (OCH 3), EDC, HOBt, DIEA, CH 2 Cl 2 or DMF

(Tetrahedron Lett, 1981, 22, 3815)

48)

MeLi, THF

49)

mCPBA, CH 2 Cl 2

fifty)

HONO, Cu2 {0, Cu (NO 3) 2, H 2 O

(J. Org. Chem. 1977, 42; 2053)

51)

R1M, solvent

52)

HONO, NaS (S) COEt, H2O

(Org. Synth. 1947, 27, 81)

53)

HSR2 or HSR4, CH 2 Cl 2

54)

i.BuOC (O) Cl, Et3N, NH3, THF

55)

R 2 R 4 NH, CH 2 Cl 2, NaBH (OAc) 3

56)

R2 R4 NH, MeOH / CH 3 CO 2 H, NaBH 3 CN

57)

R2 OH, EDC, HOBt, DIEA, CH 2 Cl 2 or DMF

58)

R2 OH, HBTU, HOBt, DIEA, CH 2 Cl 2 or DMF

59)

R 2 R 4 NH, EDC, HOBt, DIEA, CH 2 Cl 2 or DMF

60)

R 2 R 4 NH, HBTU, HOBt, DIEA, CH 2 Cl 2 or DMF

61)

POCl_ {3}, Py, CH 2 Cl 2

62)

R 2 R 4 NCO, solvent

63)

R2 OC (O) Cl, Et 3 N, solvent

64)

R 2 CO 2 H, EDC or HBTU, HOBt, DIEA, CH 2 Cl 2 or DMF

65)

R 2 X, Et 3 N, solvent

66)

(CH 3 S) 2 C = N (CN), DMF, EtOH

(J. Med. Chem. 1994, 37, 57-66)

67)

R 2 SO 2 Cl, Et 3 N, CH 2 Cl 2

68)

R 2 - or R 3 - or R 4 CHO, MeOH / CH 3 CO 2 H, NaBH 3 CN

(Synthesis 1975, 135-146)

69)

Boc (Tr) -D or L-CysOH, HBTU, HOBt, DIEA, CH 2 Cl 2 or DMF

70)

Boc (Tr) -D or L-CysH, NaBH_ {3} CN, MeOH / CH 3 CO 2 H

(Synthesis 1975, 135-146)

71)

S-Tr-N-Boc cysteinal, ClCH 2 CH 2 Cl or THF, NaBH (OAc) 3

(J. Org. Chem. 1996, 61: 3849-3862)

72)

TFA, CH 2 Cl 2, Et 3 SiH or (3: 1: 1) thioanisole / ethanedithiol / DMS

73)

TFA, CH 2 Cl 2

74)

DPPA, Et3N, toluene, HOCH_2CH_2 SiCH_ {3}

(Tetrahedron Lett. 1984, 25, 3515)

75)

TBAF, THF

76)

Base, TrSH or BnSH

77)

Base, R2 X or R2 X

78)

R 3 NH 2, MeOH / CH 3 CO 2 H, NaBH 3 CN

79)

N 2 H 4, KOH

80)

Pd 2 (dba) 3, P (o-tol) 3, RNH 2, NaOtBu, Dioxane, R1 NH2

(Tetrahedron Lett. 1996, 37, 7181-7184).

81)

Cyanamide

82)

Fmoc-Cl, bicarbonate sodium

83)

BnCOCl, sodium carbonate.

84)

AlylOCOCl, pyridine.

85)

Benzyl bromide, base.

86)

Oxalyl Chloride, DMSO.

87)

RCONH_ {2}.

88)

Carbonyldiimidazole, solvents neutral (for example, DCM, DMF, THF, toluene).

89)

Thiocarbonyldiimidazole, solvents neutral (for example, DCM, DMF, THF, toluene).

90)

Cyanogen bromide, solvents neutral (for example, DCM, DMF, THF, toluene).

91)

RCOCl, triethylamine

92)

RNHNH_ {2}, EDC

93)

RO 2 COCl, Et 3 N, DCM.

94)

MsOH, Pyridine (J. Het. Chem., 1980,607.)

95)

Base, neutral solvents (per example, DCM, toluene, THF).

96)

H 2 NOR, EDC.

97)

RCSNH_ {2}.

98)

RCOCHBrR, neutral solvents (for example, DCM, DMF, THF, toluene), (Org. Proc. Prep. Intl., 1992, 24, 127).

99)

CH 2 N 2, HCl. (Synthesis, 1993, 197).

100)

NH2NHR, neutral solvents (per example, DCM, DMF, THF, toluene).

101)

RSO 2 Cl, DMAP. (Tetrahedron Lett., 1993, 34, 2749).

102)

Et 3 N, RX. (J. Org. Chem., 1990, 55, 6037).

103)

NOCl or Cl2 (J. Org. Chem., 1990, 55, 3916).

104)

H 2 NOH, neutral solvents (for example, DCM, DMF, THF, toluene).

105)

RCCR, neutral solvents (DCM, THF, Toluene).

106)

RCHCHR, neutral solvents (DCM, THF, Toluene).

107)

H 2 NOH, HCl.

108)

Thiocarbonyldiimidazole, SiO2 or BF_ {3} OEt_ {2}. (J. Med. Chem., 1995, 39, 5228).

109)

Thiocarbonyldiimidazole, DBU or DBN. (J. Med. Chem., 1996, 39, 5228).

110)

HNO2, HCl.

111)

ClCH_2CO2 {Et} (Org. Reactions, 1959, 10, 143).

112)

Morpholine enamine (Eur. J. Med. Chem., 1982, 17, 27).

113)

RCOCHR <i> CN

114)

RCOCHR'CO2 Et

115)

Na_ {SO} {3}

116)

H 2 NCHRCO 2 Et

117)

EtO2 CCHRNCO

118)

RCNHNH_ {2}.

119)

RCOCO2H, (J. Med. Chem., 1995,38,3741).

120)

RIGHT, KOAc.

121)

2-Fluoronitrobenzene.

122)

SnCl2, EtOH, DMF.

123)

RCHO, NaBH_3 CN, HOAc.

124)

NH 3, MeOH.

125)

2,4,6-Me3 PhSO2 NH2.

126)

Et 2 NH, CH 2 Cl 2

127)

MeOC (O) Cl, Et 3 N, CH 2 Cl 2

128)

R2 NH2, EDC, HOBT, Et 3 N, CH 2 Cl 2

129)

DBU, PhCH_ {3}

130)

BocNHCH (CH 2 STr) CH 2 NH 2, EDC, HOBT, Et 3 N, CH 2 Cl 2

131)

R 2 NHCH 2 CO 2 Me, HBTU, HOBT, Et 3 N, CH 2 Cl 2

132)

BocNHCH (CH 2 STr) CH 2 OMs, LiHMDS, THF

133)

R 2 NHCH 2 C0 2 Me, NaBH (OAc) 3, ClCH 2 CH 2 Cl or THF

134)

R 2 NHCH 2 CH (OEt) 2, HBTU, HOBT, Et 3 N, CH 2 Cl 2

135)

NaBH (OAc) 3, ClCH 2 CH 2 Cl or THF, AcOH.

136)

Piperidine, DMF.

137)

Pd (Ph 3 P) 4, Bu_ {3} SnH.

138)

RC02H, EDC, HOBT, Et3N, DCM

139)

RNH2, solvents neutral

140)

RCHO, NaBH_3 CN, HOAc.

141)

RNCO, solvent.

142)

RCO 2 H, EDC or HBTU, HOBt, DIEA, CH 2 Cl 2 or DMF.

143)

RCOCl, Triethylamine

144)

RSO 2 Cl, Et 3 N, CH 2 Cl 2.

145)

SnCl2, EtOH, DMF.

146)

RNH2, EDC, HOBt, DIEA, CH 2 Cl 2 or DMF.

147)

Dibromoethane, Et3N, CH 2 Cl 2

148)

Oxalyl Chloride, solvents neutral

149)

LiOH, THF-MeOH.

150)

Carbonyldiimidazole, solvents neutral (for example, DCM, DMF, THF, toluene).

151)

RNH2, Et3N, CH 2 Cl 2.

152)

Base, RX.

153)

DBU, PhCH_ {3}

154)

DPPA, Et3N, toluene (Synthesis 1985, 220)

155)

SOCl2, cat DMF.

156)

ArH, Lewis acid (AlCl 3, SnCl 4, TiCl 4), CH 2 Cl 2.

157)

H 2 NCHRCO 2 Et, solvents neutral

158)

BocHNCHRCO2H, EDC OR HBTU, HOBt, DIEA, CH 2 Cl 2 or DMF.

159)

TFA, CH 2 Cl 2.

b. Illustrative preparation of aryl subunits

Any aryl subunit can be functionalized using a wide variety of reactions known to experts in the technique The chemistry of aromatic rings and Heteroaromatic is rich, and can only occur in the present Document a sample of useful reactions. Then you They show several illustrative examples.

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Suzuki coupling No. 1:

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twenty

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Suzuki coupling No. 2:

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twenty-one

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Stille coupling No. 1:

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22

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Stille coupling No. 2:

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2. 3

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Stille coupling No. 3

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24

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C. Illustrative preparation of substrates of coupling

The members of the general classes of coupling substrates explained above - arilestannanos, arylboronic acids, aryl triflates, and aryl halides - are available from the parent heterocycles. In general, the transformations required to prepare a substrate of Coupling are reliable and easy to scale. TO Illustrative examples are shown below.

Preparation of aryl iodide

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25

Preparation of aryl stannan

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26

Preparation of aryl triflate

27

Preparation of aryl boronic acid

28

d. Example procedures for preparing compounds object

Route one

29

Acid methyl ester 2- (2-benzyloxycarbonylamino-acetylamino-benzoic acid (one)

At a dissolution of N-? -Cbz-glycine (2.0 g, 9.56 mmol), in tetrahydrofuran (20 ml), was added 1-1'-carbonyldiimidazole (1.64 g, 10.13 mmol) followed 2 hours later by methyl anthranilate (1.28 g, 8.47 mmol). After stirring overnight, the tetrahydrofuran in vacuo and the residue was redissolved in ethyl acetate, washed with 10% aqueous hydrochloric acid (2x), aqueous saturated sodium hydrogen carbonate (2x), dried (MgSO 4) and concentrated to give an oily residue that was subjected to  column chromatography on silica gel [eluent: acetate ethyl: hexane, 30: 70, v / v? ethyl acetate] giving the title benzoate (1) (0.31 g, 11%) as a white solid:

δ (360 MHz; CDCl 3) 3.89 (s, 3H), 4.10 (d, 2H), 5.19 (s, 2H), 7.11 (t, 1H), 7.31-7.41 (m, 5H), 7.56 (t, 1H), 8.03 (d, 1H), 8.69 (d, 1H) and 11.54 (s, 1H).

Acid 2- (2-benzyloxycarbonylamino-acetylamino-benzoic acid (2)

Lithium hydroxide (55 mg, 1.32 mmol) was added in water (0.8 ml) to a solution of benzoate (1) (300 mg, 0.88 mmol) in dioxane (4 ml). After stirring overnight, the mixture is concentrated at 40 ° C to give a viscous oil, which was treated with acid 10% aqueous hydrochloric (3 ml), the emulsion being extracted resulting formed with ethyl acetate (4x). Organic phases were combined, dried (MgSO 4) and concentrated to give the Title benzoic acid (2) quantitatively as a solid White:

δ (360 MHz; CDCl 3) 3.84 (d, 2H), 5.12 (s, 2H), 7.21 (t, 1H), 7.35-7.44 (m, 5H), 7.65 (t, 1H), 8.04 (t, 1H), 8.65 (d, 1H) and 11.74 (s, 1H).

Benzyl Acid Ester [3- (4-Fluoro-phenyl) -4-oxo-3,4-dihydro-quinazolin-2-ylmethyl] -carbamic (3)

To a solution of benzoic acid (2) (180 mg, 0.55 mmol) in tetrahydrofuran (6 ml) was added 1,1'-carbonyldiimidazole (98 mg, 0.60 mmol). After a 2 h period, 4-fluoroaniline (61 mg, 0.55 mmol) in tetrahydrofuran (1.5 ml) and the mixture was stirred at 75 ° C all night. Tetrahydrofuran was removed in vacuo and the residue was dissolved again in ethyl acetate, washed with acid 10% aqueous hydrochloric, saturated sodium hydrogen carbonate aqueous, dried (MgSO4) and concentrated to give an oily residue which underwent column chromatography on silica gel [eluent: ethyl acetate: hexane, 20:80, v / v \ 60:40 v / v] giving the title quinazoline (3) (0.11 g, 50%) as a white solid:

δ (360 MHz; CDCl 3) 4.01 (d, 2H), 5.13 (s, 2H), 6.27 (s, 1H), 7.31-7.39 (m, 9H), 7.52 (t, 1H), 7.71 (d, 1H), 7.81 (t, 1H) and 8.28 (d, 1H).

2-Aminomethyl-3- (4-fluoro-phenyl) -3H-quinazolin-4-one (4)

A stirred mixture of quinazoline was evacuated (3) (63 mg, 0.16 mmol), 10% palladium on activated carbon (18 mg) and methanol (4.4 ml), using a vacuum pump and filled with hydrogen Once the starting material has been consumed, monitored by CCF analysis (thin layer chromatography), the mixture was filtered through a Celite pad that was washed with methanol (2x) and concentrated to give the title amine (4) (40 mg, 93%) as a yellow solid:

δ (360 MHz; CDCl 3) 3.50 (s, 2H), 7.25 (apparent d, 4H), 7.50 (t, 1H), 7.75-7.82 (m, 2H), and 8.28 (d, 1 H).

1- (4-Chloro-3-trifluoromethyl-phenyl) -3- [3- (4-fluoro-phenyl) -4-oxo-3,4-dihydroquinazolin-2-ylmethyl] urea (5)

To a mixture of the amine (4) (14 mg, 0.05 mmol) in chloroform, isocyanate was added 3-trifluoromethylphenyl (10 mg, 0.05 mmol). After stir overnight the solvent was removed in vacuo, triturated with hexanes, filtered and dried to give the title urea (5) (24 mg, 98%) as a white solid:

δ (360 MHz; CDCl 3) 4.08 (s, 2H), 6.52 (s, 1H), 7.16-7.38 (m, 4H), 7.48-7.53 (m, 2H), 7.59 (d, 1H), 7.66 (s, 1H), 7.75 (t, 1H), 7.81 (s, 1H) and 8.29 (d, 1H).

1- (2,6-Dichloro-pyridin-4-yl) -3- [3- (4-fluoro-phenyl) -4-oxo-3,4-dihydroquinazolin-2-ylmethyl] urea (6)

To a mixture of the amine (4) (14 mg, 0.05 mmol) in chloroform, isocyanate was added 2,6-dichloropyridyl (11 mg, 0.05 mmol). After shaking overnight, the solvent was removed in vacuo, triturated with hexanes, filtered and dried to give the title urea (6) (22 mg, 96%) as a white solid:

δ (360 MHz; CDCl 3) 4.09 (d, 2H), 6.41 (sa, 1H), 6.69 (dd, 1H), 6.75 (s, 1H), 6.87 (dd, 1H), 7.07 (t, 1H), 7.22-7.27 (m, 3H), 7.52 (t, 1H), 7.65 (d, 1H), 7.79 (t, 1H) and 8.29 (dd, 1H).

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Route 2

30

2-Ethyl-3,1- [4H] benzoxazin-one (7)

Anthranilic acid (100 g, 0.73 mol) was heated and propionic anhydride (420 ml) up to 140 ° C for 3.5 h, in a flask provided with Claisen distillation head. Be the temperature increased to 170-180 ° C, and it removed propionic acid and propionic anhydride under pressure reduced giving a brown solid, which was triturated with hexane, was filtered and dried to give the title benzoxazine (7) (115.8 g, 91%),  as an off-white solid:

δ (360 MHz; CDCl 3) 1.35 (t, 3H), 2.71 (q, 2H), 7.45-7.50 (m, 1H), 7.55 (d, 1H), 7.75-7.79 (m, 1H), 8.16 (dd, 1H).

2-Ethyl-3- (4'-fluoro-phenyl) -quinazolin-4-one (8)

A solution of benzoxacin was heated (7) (5.0 g, 28.6 mmol) and 4-fluoroaniline (3.37 g, 30.0 mmol) in chloroform (12 ml) at reflux overnight. Once I know fully consumed the benzoxacin (7) monitored by CCF, it the solvent was removed and the residual off-white solid was suspended in ethylene glycol (8 ml) to which sodium hydroxide (51) had been added mg) and the suspension was heated to 120-140 ° C in a distillation apparatus The water produced was distilled during a 5 h warm-up period, and the dark solution was left cool to room temperature overnight. The pH was adjusted to 7-8 by the addition of 3% hydrochloric acid, and the precipitate was filtered and dried to give the title quinazolinone (8) (4.42 g, 58%) as a white solid:

δ (360 MHz; CDCl 3) 1.23 (t, 3H), 2.44 (q, 2H), 7.24 (apparent d, 4H), 7.44-7.49 (m, 1H), 7.71-7.79 (m, 2H), 8.26 (dd, 1H).

2- (1'Bromoethyl) -3- (4 '' - fluoro-phenyl) -quinazolin-4-one (9)

To a stirred mixture of quinazolinone (8) (94.0 g, 0.35 mmol), anhydrous sodium acetate (35.2 g, 0.43 mol) and glacial acetic acid (448 ml) at 40 ° C under an atmosphere of nitrogen, a bromine solution (76.8 g, 0.48 mol) in glacial acetic acid (243 ml), while kept the mixture at about 40 ° C for a period of 5 h. The mixture was poured into water (5.26 l), stirred for 1 h and filter. The filtrate cake was washed with warm water (2.8 L) and dried giving the title bromide (9) (117.2 g, 96%) as a solid White:

δ (360 MHz; CDCl 3) 1.99 (d, 3H), 4.48 (q, 1H), 7.07-7.25 (m, 3H), 7.44-7.52 (m, 2H), 7.73-7.75 (m, 2H), 8.21 (d, 1H).

2 - [(1'-Methylamino) -ethyl] -3- (4 '' - fluorophenyl) -quinazolin-4-one (10)

A 8.03 molar solution of methylamine was added in ethanol (923 ml, 7.41 mol) in bromide (9) (117.0 g, 0.34 mol) and the suspension was heated to 40 ° C. After 1 h the material The starting point was fully consumed, as monitored by CCF and the solvent and excess methylamine were removed under pressure reduced The residue was suspended in dichloromethane (1.2 L), stirred for 1 h at room temperature, filtering the precipitated methylammonium hydrochloride, and washed with dichloromethane (117 ml). The combined organic phases were concentrated and dried (MgSO 4) giving the title amine (10) Quantitatively as a white solid:

δ (360 MHz; CDCl 3) 1.30 (d, 3H), 2.33 (s, 3H), 3.38 (q, 1H), 7.26-7.34 (m, 4H), 7.51-7.55 (m, 1H), 7.76-7.85 (m, 2H), 8.31 (dd, 1H).

2- {1 '- [N- (3' '- trifluoromethylphenylcarbamoyl) -N-methyl-amino] ethyl} -3- (4''fluorophenyl) -quinazolin-4-one (eleven)

To a solution of the amine (10) (18.9 g, 0.065 mol) and chloroform (150 ml) an isocyanate solution of 3-trifluoromethylphenyl (11.0 g, 0.065 mol) in chloroform (39 ml), while maintaining the temperature by below 30 ° C. The mixture was stirred at room temperature for 1 h, and the precipitate formed was filtered, washed with chloroform and dried giving the title urea (11) (20.2 g, 65%) as a solid whitish:

δ (360 MHz; CDCl 3) 1.55 (d, 3H), 2.92 (s, 3H), 5.16 (q, 1H), 7.09 (s at, 1H), 7.18-7.23 (m, 1H), 7.29-7.30 (m, 3H), 7.34-7.43 (m, 2H), 7.55-7.58 (m, 3H), 7.80-7.88 (m, 2H), 8.31 (d, 1H).

2- {1 '- [N- (2' '- methoxy-5' '- trifluoromethylphenylcarbamoyl) -N-methyl-amino] ethyl} -3- (4''fluorophenyl) -quinazolin-4-one (12)

A solution of 2-methoxy-5-trifluoromethylaniline (42 mg, 0.22 mmol) in ethyl acetate (1.2 ml) at a solution of triphosgene (80 mg, 0.27 mmol) in ethyl acetate (1.2 ml), is added catalytic amounts of activated carbon and the mixture was heated to reflux for 2 h. After cooling to room temperature, the solvent was evaporated under reduced pressure, the resulting residue in chloroform (1.2 ml). A solution was added of the amine (10) (65 mg, 0.22 mmol) in chloroform (1.2 ml) and the mixture was stirred at room temperature until consumed Totally the starting material. The solvent and the solvent were evaporated. crude product was subjected to column chromatography on gel silica [eluent: dichloromethane? ethyl acetate], isolating the title urea (12) (98 mg, 87%) as a solid White:

δ (360 MHz; CDCl 3) 1.40 (d, 3H), 2.88 (s, 3H), 3.83 (s, 3H), 5.25 (q, 1H), 6.80 (d, 1H), 6.97-7.03 (m, 2H), 7.13-7.18 (m, 3H), 7.20-7.25 (m, 1H), 7.40-7.45 (m, 1H), 7.65-7.74 (m, 2H), 8.18-8.22 (m, 2H).

N- {1- [3- (4-Fluoro-phenyl) -4-oxo-3,4-dihydro-quinazolin-2-yl] ethyl} -N-methyl-3-trifluoromethyl-benzamide (13)

To a solution of the amine (10) (50 mg, 0.17 mmol) and N, N -diisopropylethylamine (0.03 ml, 0.17 mmol) in dichloromethane (0.5 ml) was added 3-trifluoromethylbenzoyl chloride (0.03 ml, 0.19 mmol). The mixture was stirred overnight at room temperature under a nitrogen atmosphere. After 12 h, some 4-dimethylaminopyridine crystals were added, and stirring was continued for another 12 h. The mixture was quenched with water, washed with saturated aqueous sodium hydrogen carbonate, dried (MgSO4), filtered and concentrated to give the title benzamide (13) quantitatively as a white foam:

δ (360 MHz; CDCl 3) 1.53 (d, 3H), 3.16 (s, 3H), 5.36 (q, 1H), 7.24-7.36 (m, 3H), 7.45-7.56 (m, 3H), 7.64-7.82 (m, 5H), 8.29 (d a, 1H).

N- {1- [3- (4-Fluoro-phenyl) -4-oxo-3,4-dihydro-quinazolin-2-yl] ethyl} -N-methyl-3-trifluoromethyl-benzenesulfonamide (14)

To a solution of the amine (10) (50 mg, 0.17 mmol) and N, N- diiospropylethylamine (0.03 ml, 0.17 mmol) in dichloromethane (0.5 ml) was added 3-trifluoromethylphenylsulfonyl chloride (0.03 ml, 0.19 mmol) and the mixture was stirred at room temperature under a nitrogen atmosphere. After 12 h, some 4-dimethylaminopyridine crystals were added, and stirring was continued for another 12 h. The mixture was quenched with water, washed with saturated aqueous sodium hydrogen carbonate, dried (MgSO4), filtered and concentrated to give the title benzamide (14) quantitatively as a white foam:

δ (360 MHz; CDCl 3) 1.28 (d, 3H), 3.16 (s, 3H), 4.92 (q, 1H), 7.18-7.30 (m, 2H), 7.35-7.50 (m, 5H), 7.62 (d a, 1H), 7.70 (dt, 1H), 7.79 (d a, 1H), 7.87 (s a, 1H), 8.25 (dd, 1H).

2- {1-Bis- (2-methoxy-ethyl) amino] -ethyl} -3- (4-fluoro-phenyl) 3-H-quinazolin-4-one (fifteen)

A solution of bromide (9) (71.5 mg, 0.21 mmol), tetrahydrofuran (1.0 ml) and bis- (2-methoxyethyl) amine (0.33 ml, 2.26 mmol) It was heated to 70 ° C. After 6.5 hours, the reaction was concentrated. partially, and ethyl acetate (1 ml) and water (1 ml) were added stirring the mixture vigorously for 1h. The layer was separated organic, washed with water (2 x 1 ml), dried (MgSO 4), dried filtered and concentrated in vacuo to give the title amine (15) (61 mg, 74%) as an oil:

δ (360 MHz; CDCl 3) 1.35 (d, 3H), 2.51-2.56 (m, 2H), 2.74-2.83 (m, 2H), 2.98-3.11 (m, 4H), 3.12 (s, 6H), 3.79 (q, 1H), 7.13-7.27 (m, 3H), 7.45-7.54 (m, 2H), 7.74-7.76 (m, 2H), 8.26 (d, 1H).

Route 3

31

2-Ethyl-pyrido [2.3-d] [1.3] oxazin-4-one (16)

An acid suspension 2-aminonicotinic (1.0 g, 7.24 mmol) in anhydride propionic (10 ml) was heated at 120 ° C for 1 h under the atmosphere of nitrogen. The temperature was increased to 167 ° C to eliminate propionic acid and propionic anhydride under pressure reduced The resulting brown solid was triturated with hexane and dried giving the title pyrido-oxazine (16) (1.10 g, 86%) as a white solid:

δ (360 MHz; CDCl 3) 1.43 (t, 3H), 2.82 (q, 2H), 7.48 (dd, 1H), 8.53 (dd, 1H), 8.98 (d, 1H).

2-Ethyl-3- (4-fluoro-phenyl) -3H-pyrido [23-d] pyrimidin-4-one (17)

A suspension of the pyrido-oxazine (16) (0.5 g, 2.84 mmol) and 4- fluoroaniline (0.32 g, 2.84 mmol) in toluene (21 ml) at reflux All night long. The solution was concentrated to dryness. under reduced pressure and ethylene glycol (2 ml) was added and sodium hydroxide (5 mg). The mixture was heated again at 120 ° C. for 4 h, and ethylene glycol was distilled off under reduced pressure. He resulting dark brown solid underwent column chromatography on silica gel [eluent: ethyl acetate: hexane, 20:80 v / v ? 100: 0 v / v] giving pyrido-pyrimidone of the title (17) (244 mg, 32%) as a beige solid:

δ (360 MHz; CDCl 3) 1.31 (t, 3H), 2.49 (q, 2H), 7.25-7.28 (m, 4H), 7.44 (dd, 1H), 8.59 (dd, 1H), 9.00 (dd, 1H).

2- (1-Bromo-ethyl) -3- (4-fluoro-phenyl) -3H-pyrido [2.3-d] pyrimidin-4-one (18)

A quinazolinone suspension was heated (17) 850.0 g, 0.56 mmol) and anhydrous sodium acetate (56 mg) in acid glacial acetic acid (1.0 ml) up to 40 ° C and treated dropwise with a bromine solution (90 mg, 0.56 mmol) in glacial acetic acid (5 ml) Once the starting material was completely consumed by TLC analysis, the mixture was poured into water (5 ml), basified with aqueous saturated sodium hydrogen carbonate and extracted with tert-butylmethyl ether. The combined organic phases dried (MgSO 4), filtered and concentrated to give a solid that was purified by column chromatography on gel silica [eluent: ethyl acetate: hexane, 50:50, v / v] giving the title bromide (18) (166 mg, 85%) as a solid whitish

δ (360 MHz; CDCl 3) 1.21 (d, 3H), 4.58 (q, 1H), 7.15-7.34 (m, 3H), 7.51 (dd, 1H), 7.55-7.60 (m, 1H), 8.62 (dd, 1H), 9.05 (dd, 1H).

3- (4-Fluoro-phenyl) -2- (1-methylamino-ethyl) -3H-pyrido [2.3-d] pyrimidin-4-one (19)

A suspension of the bromide (18) was heated (80 mg, 0.23 mmol) and methylamine solution in 8.03 M ethanol (0.64 ml) up to 40 ° C for 3 h, and the whole night. The resulting suspension was concentrated to dryness under reduced pressure, and the resulting light brown solid was suspended in dichloromethane and filtered through a plug of silica gel giving the title amine (19) (57 mg, 83%) as a beige solid. (The raw material was taken directly for the next stage without additional purification).

1- {1- [3 [(4-Fluoro-phenyl) 4-oxo-3,4-dihydro-pyrido [2.3-d] pyrimidin-2-yl] -ethyl} -1-methyl-3- (3- trifluoromethyl-phenyl) -urea (twenty)

To a solution of the amine (19) (30 mg, 0.1 mmol) in dichloromethane (0.2 ml) was added isocyanate 3-trifluoromethyl-phenyl (19 mg, 0.1 mmol). The mixture was stirred at room temperature throughout the night, after which the precipitate was filtered, washed with dichloromethane and dried to give the title urea (20) (6 mg, 13%) as a white solid:

δ (360 MHz; CDCl 3) 2.17 (d, 3H), 3.15 (s, 3H), 5.23 (q, 1H), 7.15 (s at, 1H), 7.22-7.31 (m, 4H), 7.38 (t, 1H), 7.49 (d, 1H), 7.50-7.56 (m, 2H), 7.62 (s at, 1H), 8.62 (dd, 1H), 9.00 (d, 1H).

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Route 4

32

Acid 3-exo-tert-butoxycarbonylamino-bicyclo [2.2.1] hept-5-eno-2-exo-carboxylic (twenty-one)

An acid solution 3-exo-amino-bicyclo [2.2.1] hept-5-eno-2-exo-carboxylic (1.0 g, 6.5 mmol), 1N aqueous sodium hydroxide (6.5 ml) and dioxane (6.5 ml) was cooled in an ice bath, and dicarbonate of di-tert-butyl (3.0 g, 13.8 mmol) with continued stirring for 10 minutes at 0 ° C and 6 h at room temperature. The solvent was partially evaporated and adjusted the pH to 1-2 by adding hydrogen sulfate 1N aqueous potassium (~ 8 ml). The aqueous phase was extracted with ethyl acetate (3 x 10 ml) and the combined organic phases are dried (MgSO 4), and concentrated to give the title acid (21) (1.41 g, 85%) as a white solid:

δ (360 MHz; CDCl 3) 1.70 (s, 9H), 1.89 (m, 1H), 2.31 (m, 1H), 2.84 (m, 1H), 2.98 (m, 1H), 3.22 (m, 1H), 4.20 (m, 1H), 6.44 (m, 2H), 7.21 (m, 1H).

Tert-butyl acid ester [3-exo- (4-fluoro-phenylcarbamoyl) -bicyclo [2.2.1] hept-5-eno-2-exo-il] -carbamic (22)

A solution of the acid (21) (1.4 g, 5.5 mmol) in anhydrous tetrahydrofuran (20 ml) it was cooled to -10 ° C and treated dropwise with triethylamine (0.77 ml, 5.5 mmol) followed by isobutyl chloroformate (0.72 ml, 5.5 mmol). The suspension resulting was stirred for 10 minutes at -10 ° C before adding a 4-fluoroaniline solution (0.53 ml, 5.5 mmol) in anhydrous tetrahydrofuran (5 ml), keeping the temperature between -8 and -17 ° C for 5 h and allowing the mixture to temper at room temperature all night. Triethylamine Hydrochloride it was filtered off, washed with tetrahydrofuran, and the solvent evaporated to dryness, washing the solids resulting with water (20 ml) and drying giving the acid ester Carbamic title (22) quantitatively as a solid White:

δ (360 MHz; CDCl 3) 1.24 (s, 9H), 1.66 (d, 1H), 2.06 (d, 1H), 2.45 (d, 1H), 2.71 (s at, 1H), 3.08 (s at, 1H), 3.93-4.06 (m, 1H), 4.96 (d, 1H), 6.18-6.24 (m, 2H), 6.94-7.02 (m, 2H), 7.48-7.50 (m, 2H), 7.98 (s at, 1H).

(4-fluoro-phenyl) -amide of the acid 3-exo-amino-bicyclo [2.2.1] hept-5-eno-2-exo-carboxylic (2. 3)

To an ester solution of carbamic acid (22) (2.24 g) in dichloromethane (50 ml) acid was added trifluoroacetic acid (5 ml). The solution was stirred at temperature ambient for 4 h before water (50 ml) was added. Be The aqueous phase was separated and washed with dichloromethane (25 ml), filtered and was basified with 2N sodium hydroxide. The basified aqueous phase is extracted with dichloromethane (2 x 25 ml), and the organic phases combined, washed with brine (10 ml), dried (MgSO4), and concentrated giving the title amine (23) (870 mg, (64% after 2 stages from (21)) as a solid.

δ (360 MHz; CDCl 3) 1.59 (d, 1H), 1.71 (d a, 2H), 2.13 (d, 1H), 2.37 (d, 1H), 2.59 (s a, 1H), 3.09 (s a, 1H), 3.24 (d, 1H), 6.20 (s at, 2H), 6.99 (t, 2H), 7.47-7.51 (m, 2H), 8.54 (s a, 1H).

2-Ethyl-3- (4-fluoro-phenyl) -3H-pyrimidin-4-one (24)

A solution of the amine (23) (43 mg, 0.17 mmol) in triethylortopropionate (0.5 ml, 2.38 mmol) was heated to 100 ° C for 26 h, after which the excess of solvent and volatile coproducts under reduced pressure giving the Title pyrimidone (24) (41 mg, quant.) as a solid crystalline:

δ (360 MHz; CDCl 3) 1.17 (t, 3H), 2.37 (q, 2H), 6.45 (d, 1H), 7.17-7.27 (m, 4H), 7.93 (d, 1 HOUR).

2- (1-Bromo-ethyl) -3- (4-fluoro-phenyl) -3H-pyrimidin-4-one (25)

A solution of pyrimidone (24) (150 mg, 0.69 mmol) and sodium acetate (140 mg) in glacial acetic acid (1.5 ml) was warmed to 40 ° C and treated dropwise with a solution of preformed bromine (0.7 ml of bromine in 10 ml of acetic acid glacial) (0.55 ml, 0.69 mmol). After 2 h, water (20 ml) was added to the mixture that was subsequently basified with potassium carbonate, and extracted with dichloromethane (2 x 10 ml). Organic phases combined they were washed with water (10 ml) dried (MgSO4), and dried concentrated giving the title bromide (25) (196 mg, 96%) as a pale brown oil:

δ (360 MHz; CDCl 3) 1.95 (d, 3H), 4.46 (q, 1H), 6.50 (d, 1H), 7.12-7.16 (m, 1H), 7.20-7.28 (m, 2H), 7.43-7.50 (m, 1H), 8.81 (d, 1H).

3- (4-Fluoro-phenyl) -2- (1-methylamino-ethyl) -3H-pyrimidin-4-one (26)

The bromide (25) (190 mg, 0.64 mmol) was dissolved in a 33% solution of methylamine in ethanol (5 ml) and stirred at room temperature. After 3.5 h, the solvent and the solvent were evaporated. excess methylamine under reduced pressure and the residue was partitioned between water (10 ml) and dichloromethane (10 ml). The organic layer was washed again with water (10 ml), dried (MgSO4), and concentrated giving the title amine (26) (120 mg, 76%) as an oil yellow:

δ (360 MHz; CDCl 3) 1.20 (d, 3H), 2.23 (s, 3H), 3.26 (q, 1H), 6.44 (d, 1H), 7.16-7.33 (m, 4H), 7.98 (d, 1H).

1- {1- [1 (4-Fluoro-phenyl) -6-oxo-1,6-dihydro-pyrimidin-2-yl] -ethyl} -1-methyl-3- (3-trifluoromethyl-phenyl) -urea (27)

The amine (26) (12 mg, 0.05 mmol) was dissolved and 3-Trifluoromethylphenyl isocyanate (7.5 L, 0.05 mmol) in chloroform (0.2 ml) and stirred at room temperature. After the complete conversion of the starting material by analysis by TLC, the solvent was removed and the resulting residue was subjected to column chromatography on silica gel [eluent: dichloromethane] giving the title urea (27) (14 mg, quant.) as a solid:

δ (360 MHz; CDCl 3) 1.61 (m, 3H), 2.87 (s, 3H), 5.04 (m, 1H), 6.41 (m, 1H), 6.64 (s, 1H), 7.11-7.48 (m, 8H), 7.86 (m, 1H).

Nitro reduction to anilines

33

2- {1 '- [N-3' '- (amino-carbamoyl) -N-methyl-amino] ethyl} -3- (4' '- fluorophenyl) -quinazolin-4-one (29)

A solution of 2- {1 '- [N- (3''nitro-carbamoyl) -N-methyl-amino] ethyl} -3- (4''- fluorophenyl) -quinazolin-4-one (28)
(168 mg, 0.036 mmol), dichloromethane (5 ml) and ethyl acetate (5 ml) was stirred under a hydrogen atmosphere in the presence of 10% activated palladium on carbon until the starting material was completely consumed. The mixture was filtered through Celite and concentrated to dryness to give the title aniline (29) (100 mg, 65%):

δ (360 MHz; CDCl 3) 1.48 (d, 3H), 2.88 (s, 3H), 5.17 (q, 1H), 6.39 (s at, 1H), 6.50 (s at, 1H), 6.92 (s at, 1H), 6.60 (s at, 1H), 7.02 (t, 1H), 7.15-7.33 (m, 4H), 7.49-7.55 (m, 1H), 7.75-7.82 (m, 2H), 8.27 (d, 1H).

An in situ method was developed to derivatize the pendant amine, as exemplified in the following protocol:

3. 4

1- {1- [3 (Fluoro-phenyl) -4-oxo-3,4-dihydro-quinazolin-2-yl] -ethyl} -1-methyl-3- (3-trifluoromethanesulfonyl-phenyl) -urea (31)

A solution of triphosgene (160 mg, 0.54 mmol) and ethyl acetate (2.5 ml) was added to a solution of (3-aminophenyl) trifluoromethyl sulfone (100 mg, 0.44 mmol) and ethyl acetate (2.5 ml). After stirring for 5 minutes the mixture was refluxed until it was until transparent. The mixture was concentrated and dissolved again in chloroform (2.5 ml) until a solution of the amine was added (30) (130 mg, 0.44 mmol) in chloroform (2.5 ml). Once all the starting material had been consumed by TLC, the solvent was removed in vacuo and subjected to column chromatography on gel silica using dichloromethane and then ethyl acetate as eluent giving the title urea (31) (226 mg, 94%) as a solid.

δ (360 MHz; CDCl 3) 1.43 (d, 3H), 2.88 (s, 3H), 4.98 (s at, 1H), 7.08-7.14 (m, 1H), 7.16-7.31 (m, 3H), 7.45-7.54 (m, 3H), 7.60 (d, 1H), 7.69-7.80 (m, 3H), 7.96 (d, 1H) and 8.22 (d, 1 H).

1- {1- [3 (4-Fluoro-phenyl) -4-oxo-3,4-dihydro-quinazolin-2-yl] -ethyl} -3- (3- trifluoromethyl-4-nitro-phenyl) -1-methyl-urea (32)

A solution of triphosgene (80 mg, 0.27 mmol) and ethyl acetate (1.2 ml) was added to a solution of (4-nitro-3-trifluoromethylaniline  (45 mg, 0.22 mmol) and ethyl acetate (1.2 ml). After stirring for 5 minutes the mixture was refluxed until it was until transparent. The mixture was concentrated and dissolved again in chloroform (1.2 ml) until a solution of the amine was added (30) (65 mg, 0.22 mmol) in chloroform (1.2 ml). Once all the starting material had been consumed by TLC, the solvent was removed in vacuo and subjected to column chromatography on gel silica using dichloromethane and then ethyl acetate as eluent giving the title urea (32) (112 mg, 97%) as a solid.

δ (360 MHz; CDCl 3) 1.45 (d, 3H), 2.80 (s, 3H), 4.90 (s at, 1H), 7.05- 7.25 (m, 4H), 7.48 (t, 1H), 7.63-7.79 (m, 4H), 7.86 (d, 1H), and 8.19 (d, 1H).

1- {1- [3 (4-Fluoro-phenyl) -4-oxo-3,4-dihydro-quinazolin-2-yl] -ethyl} -3- (3- trifluoromethyl-4- (4-methyl) -piperazin-i-yl) -phenyl) -1-methyl-urea (33)

A solution of triphosgene (80 mg, 0.27 mmol) and ethyl acetate (1.2 ml) was added to a solution of 4- (N-methylpiperazine) -3-trifluoromethyl aniline (56 mg, 0.22 mmol) and ethyl acetate (1.2 ml). After shaking for 5 minutes, the mixture was refluxed until it was transparent. The mixture was concentrated and dissolved again in chloroform (1.2 ml) until a solution of the amine was added (30) (65 mg, 0.22 mmol) in chloroform (1.2 ml). Once all the starting material had been consumed by TLC, the solvent was removed in vacuo and subjected to column chromatography on gel silica using ethyl acetate and then methanol at 5-10% in dichloromethane as eluent giving urea of the title (33) (25 mg, 20%) as a solid.

δ (360 MHz; CDCl 3) 1.38 (d, 3H), 1.97 (s at, 2H), 2.26 (s, 3H), 2.47 (s at, 2H), 2.72-2.86 (m, 7H), 4.92-5.05 (m, 1H), 6.80-6.93 (s at, 1H), 7.08 (t, 1H), 7.12-7.27 (m, 4H), 7.34 (s, 1H) 7.41 (t, 2H), 7.63-7.74 (m, 2H) and 8.16 (d, 1H).

All references cited above are incorporated herein by reference herein document.

Equivalent

Those skilled in the art will recognize, or be able to establish, using only experimentation routine, many equivalent to the specific embodiments of the invention described herein. Such equivalents are It is intended to be encompassed by the following claims.

Claims (32)

1. Use of a compound for the preparation of a pharmaceutical composition to inhibit hyperproliferation of a cell, in which the compound is an organic molecule represented by the general formula (II): 35 in which, to the extent that the valence and stability what allow, R 1 and R 2, independently for each appearance, represent H, C 1 -C 10 alkyl, aryl (substituted or unsubstituted), aralkyl (substituted or not substituted), heteroaryl (substituted or unsubstituted), or heteroaralkyl (substituted or unsubstituted); L, independently for each situation, is absent or represents -alkenyl-, alkynyl-, - (CH 2) n O (CH 2) p -,
- (CH 2) n NR 2 (CH 2) p -, - (CH 2) n S (CH 2) p - , - (CH 2) n alkenyl (CH 2) p -, - (CH 2) n alkynyl (CH 2) p -, - O (CH 2) n -, -NR 2 (CH 2) n - or -S (CH 2) n -; X is selected from -N (R 8) -, -O-, -S-, -Se- -N = N-, -ON = CH-, (R 8) N-N (R 8) -, -ON (R 8) -, a heterocycle, or a direct link between L e Y; And select from -C (= O) -, -C (= S) -, -S (O2) -, -S (O) -, -C (= NCN) -, -P (= O) (OR_2) -, a heteroaromatic group, or a direct link between X and Z; Z is selected from -N (R 8) -, -O-, -S-, -Se- -N = N-, -ON = CH-, R 8 N-NR 8 -, -ONR 8 -, a heterocycle, or a direct link between Y and L; R_ {8}, independently for each situation, represents H, C 1 -C 10 alkyl, aryl (substituted or unsubstituted), aralkyl (substituted or not substituted), heteroaryl (substituted or unsubstituted), or heteroaralkyl (substituted or unsubstituted), or two R 8 taken together they form a ring of 4 to 8 members, together with the atoms to those that are united, whose ring that can include one or more carbonyls; W represents a benzene or pyridonic ring, substituted or unsubstituted, attached to the pyrimidone ring; p represents independently for each situation, an integer from 0 to 10; and n, individually for each situation, represents an integer from 0 to 10, in which the term "heteroaryl" or "heteroaralkyl" refers to an aryl group having up to 4 heteroatoms and in which the term "substituted" refers to a substituent selected from halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, moieties aromatic or heteroaromatic, -CF3, or -CN. 2. Use according to claim 1, wherein the cell has a hedgehog function gain phenotype. 3. Use according to claim 1 or 2, wherein R1 represents an aryl or heteroaryl group substituted or not replaced. 4. Use according to claim 1-3, in which X-Y-Z taken together they represent a urea or an amide. 5. Use according to claim 1-3, in which W is a substituted benzene ring or not replaced 6. Use according to claim 1-3, in which X represents diazacarbocycle. 7. Use according to claim 1-3, in which R2 represents an aryl group or heteroaryl, substituted or unsubstituted. 8. Use according to claim 1-3, in which R_ {8}, independently for each situation, is selected from H and alkyl C_ {1} -C_ {10}. 9. Use according to claim 1-3, in which X is selected from -N (R 8) -, -O-, -S- and a direct link; And it is selected of -C (= O) -, -C (= S) - and -S (O2) -; and Z is selected from -N (R_ {8}) -, -O-, -S- and a direct link, so that at minus one of X and Z be present. 10. Use according to claim 1-3, in which at least one of X and Z is Present. 11. Use according to claim 1-3, in which Y is selected from -C (= O) -, -C (= S) - and -S (O_ {2}) -. 12. Use according to claim 1 or 2, wherein the compound inhibits hedgehog- mediated signal transduction with an ED50 of 1 mM or less. 13. Use according to claim 1 or 2, wherein the compound inhibits hedgehog- mediated signal transduction with an ED50 of 1 µM or less. 14. Use according to claim 1 or 2, wherein the compound inhibits hedgehog- mediated signal transduction with an ED50 of 1 nM or less. 15. Use according to claim 1 or 2, wherein the compound has to be administered as part of an application Therapeutic or cosmetic. 16. Use according to claim 15, wherein the therapeutic or cosmetic application is selected from the regulation of neural tissues, bone formation and repair and cartilage, spermatogenesis regulation, regulation of smooth muscle, regulation of the lung, liver and other organs that come from the primitive intestine, the regulation of function hematopoietic or the regulation of skin growth and hair. 17. Use according to any of the claims above, in which Y is selected from -C (= O) and -C (= S) -. 18. Use according to any of the claims above, in which Z is selected from -N (R 8) -, -O-, -S- and a direct link. 19. Use according to any of the claims above, in which R2 represents a lower alkyl, group aryl or heteroaryl, substituted or unsubstituted. 20. Use according to any of the claims above, in which R_ {8}, independently for each situation, is selected from H and alkyl C_ {1} -C_ {10}. 21. Use according to any of the claims above, in which L adjacent to R1 is absent, and L adjacent to X is a substituted or unsubstituted methylene. 22. Use according to any of the claims above, in which p, individually for each situation, represents an integer in the range of 0 to 3. 23. Use according to any of the claims above, in which n, individually for each situation, represents an integer in the range of 0 to 5. 24. Use according to any of the claims above, in which R2 represents an aryl group or heteroaryl, substituted or unsubstituted. 25. Use according to any of the claims above, in which at least one of X and Z is present. 26. Use according to any of the claims above, where Z is selected from -N (R 8) -, -O- and -S-. 27. Use of a compound for the preparation of a pharmaceutical composition for the treatment of cancer, in the that the compound is an organic molecule represented by the general formula (II), as defined in any of the previous claims. 28. Use according to claim 27, wherein said cancer is basal cell carcinoma. 29. Use according to claim 28, wherein said basal cell carcinoma exists in a clinical form or selected histological nodular-ulcerative, Superficial, pigmented, morphea type, fibroepithelioma and syndrome nevoid 30. Use according to claim 27, wherein said cancer is: squamous cell carcinoma (eg, squamous cell carcinoma), carcinosarcoma, adenocystic carcinoma, epidermoid carcinoma, nasopharyngeal carcinoma, renal cell carcinoma, papilloma, epidermoid cyst, pancreatic cancer, tumors related to Gorlin syndrome (for example, medulloblastoma, meningioma, etc.), tumors evidenced in mice deficient in pct (for example, hemangioma, rhabdomyosarcoma, etc.), tumors resulting from the amplification of gli- 1 (e.g., glioblastoma, sarcoma, etc.), TRC8-related tumors, a ptc homologue (e.g., renal carcinoma, thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone cancer, etc.), Shh- induced tumors (e.g., lung cancer, chondrosarcomas, etc.), breast cancer, urogenital cancer (e.g., kidney, bladder, ureter, prostate, etc.), adrenal cancer, gastr cancer gastrointestinal (for example, stomach, intestine, etc.), CNS primary malignant neuroectodermal tumors (for example, malignant medulloblastoma), malignant gliomas, meningiomas, neuroectodermal tumors, ependymomas, pineoblastoma or keratoacanthoma. 31. Use of a compound for the preparation of a pharmaceutical composition to inhibit hair growth not desired, in which the compound is an organic molecule represented by the general formula (II), as defined in any of the preceding claims. 32. Use according to claim 27, wherein the Composition is formulated for topical administration.